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Sci Adv
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2375-2548
American Association for the Advancement of Science

adp0334
10.1126/sciadv.adp0334
Research Article
Biomedicine and Life Sciences
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Chemistry
Chemistry
Development of miRNA-based PROTACs targeting Lin28 for breast cancer therapy
miRNA-based PROTACs as efficient Lin28A degraders
https://orcid.org/0009-0003-5858-2699
Xu Jianfei Formal analysis Investigation Methodology Validation Visualization Writing - original draft †
Zhao Xiaoran Formal analysis Investigation Methodology Validation Visualization Writing - original draft †
Liang Xingxing Formal analysis Investigation Validation
Guo Dongyang Formal analysis Investigation Validation
Wang Jing Funding acquisition Writing - review & editing
https://orcid.org/0000-0001-6103-9580
Wang Qian Funding acquisition Investigation Methodology Resources Validation Writing - review & editing *
https://orcid.org/0000-0002-9959-1167
Tang Xinjing Conceptualization Data curation Funding acquisition Methodology Project administration Supervision Writing - original draft Writing - review & editing *
State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center and School of Pharmaceutical Sciences, Peking University, Xueyuan Rd, Beijing 100191, China.
* Corresponding author. Email: xinjingt@bjmu.edu.cn (X.T.); qian.wang@bjmu.edu.cn (Q.W.)
† These authors contributed equally to this work.

20 9 2024
18 9 2024
10 38 eadp033404 3 2024
09 8 2024
Copyright © 2024 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).
2024
The Authors
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Lin28, a highly conserved carcinogenic protein, plays an important role in the generation of cancer stem cells, contributing to the unfavorable prognosis of cancer patients. This RNA binding protein specifically binds to pri/pre-microRNA (miRNA) lethal-7 (let-7), impeding its miRNA maturation. The reduced expression of tumor suppressor miRNA let-7 fosters development and progression-related traits such as proliferation, invasion, metastasis, and drug resistance. We report a series of miRNA-based Lin28A-miRNA proteolysis-targeting chimeras (Lin28A-miRNA-PROTACs) designed to efficiently degrade Lin28A through a ubiquitin-proteasome–dependent mechanism, resulting in up-regulation of mature let-7 family. The augmented levels of matured let-7 miRNAs further exert inhibitory effects on cancer cell proliferation and migration, and increase its sensitivity to chemotherapy. In a mouse ectopic tumor model, Lin28A-miRNA-PROTAC demonstrates a substantial efficacy in inhibiting tumor growth. When combined with tamoxifen, the tumors exhibit gradual regression. This study displays an effective miRNA-based PROTACs to degrade Lin28A and inhibit tumor growth, providing a promising therapeutic avenue for cancer treatment with miRNA-based therapy.

miRNA-based PROTACs induce efficient Lin28A degradation, boosting up-regulation of mature let-7 family for breast cancer therapy.

http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 22277003 http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 81821004 http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 22077005 http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 22107004 http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 22077008 National key R&D program of China 2022YFC2303700 License OptionCC BY-NC
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pmcINTRODUCTION

Breast cancer, comprising ~10% of all malignant tumors in women, poses a major health challenge (1). Current treatment modalities, including surgery, hormone therapy, radiotherapy, and chemotherapy, have shown efficacy, but issues such as drug resistance, recurrence, and metastasis still persist, leading to suboptimal prognosis for patients (2–4). Thus, new therapeutic avenues or their combination therapy is still urgently in need.

MicroRNAs (miRNAs) are single-stranded endogenous RNAs that function as crucial endogenous regulators of gene expression (5, 6). miRNAs promote the suppression of particular mRNA sequences through the process of RNA interference (RNAi) (7) or interact with their target proteins and disturb protein functions. Over the last three decades, miRNAs have emerged as key players in cancer development (8). While miRNAs hold promise as diagnostic tools and therapeutic targets, especially onco-miRNAs and tumor suppressor miRNAs, their clinical translation remains in the early stages (9–11). No miRNA therapies have currently entered phase III clinical trials (12). This necessitates researchers to design more suitable strategies for miRNA therapies. Lethal-7 (let-7) family, one of the earliest discovered miRNAs (13), acts as a tumor suppressor by targeting multiple genes. Unfortunately, the low expression level of let-7 family miRNAs in cancer cells, particularly cancer stem cells, hampers their antitumor effects (14). Lin28 (Lin28A/B), a conserved RNA binding protein (RBP) and one of the four pluripotency factors, plays a crucial role in the regulation of cell growth and differentiation, metabolism, and cell pluripotency (15, 16). As an RBP, Lin28 primarily binds to the terminal loops (TLs) of let-7 precursors (17) and negatively regulates let-7 processing (18). The uridylyl transferase (TUTase) TUT4/7 is recruited to pre-let-7, and the resulting lin28-TUT4/7 complex promotes the uridylation of the 3′ terminal of pre-let-7, thereby inhibiting Dicer processing. Subsequently, uridylated pre-let-7 is degraded by ribonuclease DIS3L2 (19, 20), resulting in the down-regulation of let-7 expression levels. In recent years, more and more research on Lin28 leads to the understanding of its role in tumorigenesis (21), embryonic development (22), and cell metabolism (23). Lin28 down-regulation was found to increase the susceptibility of lung cancer radiotherapy and breast cancer chemotherapy (24, 25). The Lin28/let-7 pathway contributed to the development and progression of cancer-related phenotypes, such as proliferation, invasion, metastasis, protumor inflammation, and angiogenesis (16, 26). Thus, degrading the Lin28 level to restore sustained high expression of let-7 is proposed as a promising therapeutic approach.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules composed of a target protein ligand, a linker, and an E3 ubiquitin ligase ligand, which recruit E3 ubiquitin ligases to degrade target proteins via the ubiquitin-proteasome pathway (27–29). PROTACs offers a versatile strategy to induce target protein degradation and have progressed to the clinical trials (30, 31). Recently, oligonucleotide-based PROTACs (32–34) have gained attention, particularly for degrading transcription factors (TFs) and RBPs (35–37). However, TFs are usually involved in many gene expression, which leads to the unfavorable specificity and affinities of oligonucleotides/TFs and requires high concentration of oligonucleotide-based PROTACs (35, 36). Here, we developed a series of miRNA-based PROTACs targeting Lin28A for breast cancer treatment. Lin28A-miRNA-PROTAC combines Lin28A-binding pre-miRNA (pre-let-7f-1) with E3 ubiquitin ligase ligands (pomalidomide or VH032) (Fig. 1A). This design brings Lin28A and E3 ubiquitin ligase close to each other and facilitates Lin28A degradation, increasing the tumor suppressor miRNA let-7 level, inducing cancer cell apoptosis, inhibiting tumor growth, and increasing the sensitivity to chemotherapy. Further combined with tamoxifen (TAM), the treatment regimen successfully leads to the tumor regression in breast tumor mice model. Our findings offer a promising avenue for enhancing breast cancer treatment through targeted Lin28A degradation with miRNA-based therapy.

Fig. 1. Design and synthesis of Lin28A-miRNA-PROTAC.

(A) Lin28A-miRNA-PROTAC strategy mediates the degradation of endogenous protein Lin28A. (B) Schematic representation of human Lin28A. (C) Schematic illustration of the coupling reaction between NHS-modified pomalidomide or VH032 with various linkers and amino-modified pre-let-7f-1 for the production of Lin28A-miRNA-PROTAC.

RESULTS

Design and synthesis of Lin28A-miRNA-PROTAC

Pre-let-7 family miRNAs serve as the natural ligands for the potential anticancer target Lin28A, consisting of two distinct sequences that bind to the N-terminal cold shock domain (CSD) and the C-terminal zinc finger domain (ZKD) of Lin28A, respectively. Structural and biochemical studies have demonstrated that the CSD and ZKD domains of Lin28A bind to the GNGAY and GGAG motifs in the pre-let-7 TL (Fig. 1B) (38). For the construction of miRNA-based PROTACs (Lin28A-miRNA-PROTAC), we carefully selected oligonucleotide sequences targeting the CSD or ZKD region as well as the full length of pre-let-7f-1 (38–40), due to their high affinity to Lin28A protein, as outlined in table S1. To facilitate the ubiquitination of Lin28A, two E3 ubiquitin ligase ligands (pomalidomide and VH032) were individually conjugated to the Lin28A binding oligonucleotide sequences. These ligands are adept at hijacking Cerablon (CRBN) and von Hippel–Lindau (VHL) E3 ubiquitin ligases, respectively (table S2). In addition, diverse linkers (table S2) were also synthesized to connect the oligonucleotide sequences and E3 ubiquitin ligase ligands (outlined in the Supplementary Materials, Synthetic Procedures and Characterization, schemes S1 and S2). The final Lin28A-miRNA-PROTACs were obtained by merging the two moieties through the cross-linking of amino groups and N-hydroxysuccinimide (NHS) active esters (Fig. 1C), followed by high-performance liquid chromatography (HPLC) purification and mass spectrometry (MS) characterization, as detailed in table S3.

Degradation of Lin28A by Lin28A-miRNA-PROTAC in T47D cells

Ductal carcinoma stands out as the most common pathological type of breast cancer, accounting for approximately 70% of all breast cancer cases. Women diagnosed with ductal carcinoma in situ often confront the risk of developing a second primary breast cancer, and unfortunately, some succumb to the disease. Furthermore, the application of radiation therapy to prevent cancer recurrence has not shown a substantial reduction in mortality. Consequently, achieving a complete cure for ductal carcinoma of the breast poses a considerable challenge, as it cannot be fundamentally eradicated solely through the administration of chemotherapy drugs (41, 42). T47D cells with notably high level of Lin28A expression level were selected for subsequent degradation and antitumor experimental studies.

Before transfection, the stability of nonmodified and modified pre-let-7f-1 was evaluated. As shown in fig. S1, nonmodified pre-let-7f-1 was almost completely digested in 12 hours at the presence of the cell lysate, while more than 25% modified pre-let-7f-1 remained undegraded even at 48 hours. Following the transfection of Lin28A-miRNA-PROTACs into T47D cells, the cells were collected and lysed after 48 hours. Then, the endogenous Lin28A protein level was then assessed using Western blotting (WB). In general, under the same conditions, the degradation efficiency of PROTACs with pre-let-7f-1 sequences targeting Lin28A protein and its CSD region was notably greater than that of those targeting the ZKD region of Lin28A (Fig. 2, A and B, and fig. S2). This discrepancy is likely due to the different affinity between oligonucleotides and Lin28A protein (38). Further, by constructing mutated P7f-1-FL-P4-C derivatives, it was found that single mutation of the binding motif between P7f-1-FL-P4-C and Lin28A CSD region severely weakened its ability to degrade Lin28A. Removing the loop region of P7f-1-FL-P4-C also led to the loss of binding ability to Lin28A. However, only single mutation of the binding motif between P7f-1-FL-P4-C and Lin28A ZKD region alone had no obvious effect on Lin28A degradation. These results indicated that the binding motif of P7f-1-FL-P4-C to the CSD region of Lin28A and the loop region of P7f-1-FL-P4-C played a crucial role in its binding to Lin28A protein and its following degradation (fig. S3).

Fig. 2. Degradation of Lin28A.

(A) PROTACs derived from the P7f-1-FL sequence resulted in the degradation of Lin28A in T47D cells. T47D cells were treated with 100 nM P7f-1-FL-R1-C or P7f-1-FL-R2-V for 48 hours. (B) PROTACs derived from the P7f-1-CSD sequence resulted in the degradation of Lin28A in T47D cells. T47D cells were treated with 100 nM P7f-1-CSD-R1-C or P7f-1-CSD-R2-V for 48 hours. (C) Concentration effect of P7f-1-FL-P4-C on the degradation of Lin28A in T47D cells. T47D cells were treated with P7f-1-FL-P4-C of different concentrations at 48 hours. (D) Time effect of P7f-1-FL-P4-C on the degradation of Lin28A in T47D cells. T47D cells were treated with 100 nM P7f-1-FL-P4-C for different time. (E) Control experiments on the degradation of Lin28A in T47D cells. T47D cells were treated with 100 nM P7f-1-FL-P4-C (P7f-1-FL-ORN, P7f-1-FL-ORN + Pomalidomide, Pomalidomide) for 48 hours. (F) P7f-1-FL-P4-C induces ubiquitination of Lin28A protein in T47D cells. T47D cells were transfected with 100 nM P7f-1-FL-P4-C and His-ub. Cells were collected after 24 hours for Lin28A ubiquitination analysis. (G) Effect of MG132 proteasome inhibition on the degradation of Lin28A by P7f-1-FL-P4-C in T47D cells. T47D cells were treated with 100 nM P7f-1-FL-P4-C for 48 hours in the presence or absence of MG132 (20 μM). Values are means ± SEM with n = 3. P values were calculated with one-way ANOVA test. ns, not significant; **P < 0.01; ****P < 0.0001.

Linkers and E3 ubiquitin ligase ligands of Lin28A-miRNA-PROTACs were also screened for Lin28A degradation. As shown in Fig. 2 (A and B), the linker length and types had a substantial effect on Lin28A levels. When the E3 ubiquitin ligase ligand was pomalidomide, most Lin28A-miRNA-PROTACs with a carbon chain linker (Cx-C) demonstrated higher efficiencies in inducing Lin28A degradation. The polyethylene glycol (PEG) linker (Px-C) demonstrates the best suitability for Lin28A-miRNA-PROTACs targeting the full-length (FL) region of Lin28A. The extra linker length with 12 atoms (C12 and P4) in Lin28A-miRNA-PROTACs displayed a slightly higher Lin28A degradation that is more obvious when E3 ubiquitin ligase ligand was VH032 (Fig. 2A). As shown in Fig. 2B and fig. S2, four Lin28A-miRNA-PROTACs with CSD sequences and CRBN ligand maintained the ability to induce Lin28A degradation and P7f-1-CSD-C6-C with C6 linker displayed as the more efficient Lin28A degrader. Among Lin28A-miRNA-PROTACs with CSD sequences and VH032 ligand, P7f-1-CSD-C12-V with C12 still showed the best Lin28 degrader. However, most of Lin28A-miRNA-PROTACs with ZKD oligonucleotide sequences lost their protein degradation ability (fig. S2). These results confirmed that both linkers and E3 ligases played a vital role in target protein degradation. Thus, we selected P7f-1-FL-P4-C as the potential Lin28A-miRNA-PROTAC for further investigation.

The dose and time dependence of Lin28A degradation with P7f-1-FL-P4-C were evaluated. With the concentration increasing from 25 to 100 nM, Lin28A levels decreased to 50%, 25%, and 18% (Fig. 2C). Fixing the concentration of P7f-1-FL-P4-C at 100 nM, Lin28A expression levels were evaluated at different time. Lin28A was substantially degraded at 12 hours after transfection and achieved its maximum degradation at 48 hours after transfection (Fig. 2D), although its mRNA level increased. This indicates that miRNA-PROTAC P7f-1-FL-P4-C did not directly affect Lin28A transcription and there may exist a cellular compensation mechanism leading to the increase of mRNA level (fig. S4). To ascertain whether the degradation of Lin28A is due to Lin28A ubiquitination by Lin28A-miRNA-PROTAC rather than the effect of pre-let-7 or pomalidomide, we treated T47D cells with P7f-1-FL-ORN (pre-let-7f-1-FL) and pomalidomide alone or in physical combination. However, neither of them could effectively degrade Lin28A (Fig. 2E). P7f-1-FL-ORN and P7f-1-FL-ORN physically mixed with pomalidomide could moderately decrease the Lin28A level, and pomalidomide itself did not exhibit any degradation activity for Lin28A. In contrast, P7f-1-FL-P4-C induced a substantial degradation of Lin28A. Meanwhile, coimmunoprecipitation result revealed that miRNA-PROTAC P7f-1-FL-P4-C promoted the formation of Lin28A/P7f-1-FL-P4-C/CRBN ternary complex (fig. S5). Further ubiquitination experiments confirmed that P7f-1-FL-P4-C treatment increased an obvious polyubiquitination of Lin28A in T47D cells (Fig. 2F). Additionally, the degradation of endogenous Lin28A by P7f-1-FL-P4-C could be blocked by cotreatment with the proteasome inhibitor MG132 or the CRBN small interfering RNA (siRNA) (Fig. 2G and fig. S6). Under the treatment of MG132 (20 μM) or siCRBN (100 or 200 nM), the expression level of Lin28A recovered from about 18% to about 60%. These results together indicate that P7f-1-FL-P4-C degrades Lin28A in a ubiquitin-proteasome–dependent manner. In an unbiased global proteomic study using quantitative MS, we found that P7f-1-FL-P4-C led to the substantial degradation of Lin28A. Proteins associated with the Lin28A/let-7 pathway, such as c-Myc and KRAS, were also substantially down-regulated. Moreover, treatment with miRNA-PROTAC P7f-1-FL-P4-C also resulted in a substantial increase of many immune-related factors, such as TRI22, RSAD2, MX1, ISG15, RIGI, OAS2, IFIH, IFIT, and TRIM. This indicates that the miRNA-PROTAC treatment up-regulated the level of mature let-7 family that induced the effective immune responses in tumor cells (fig. S7). In addition, two regulatory factors (HNRPC and RALY) in miRNA processing were also substantially up-regulated with P7f-1-FL-P4-C treatment compared with pre-let-7f-1.

Up-regulation of mature let-7 family miRNA induced by Lin28A-miRNA-PROTAC

Following the successful degradation of Lin28A, the expression of the tumor suppressor let-7 family mature miRNAs in T47D cells were also assessed. Upon degradation of Lin28A by P7f-1-FL-P4-C, the expression of all mature Let-7 family miRNAs (7a to 7i) was up-regulated by more than 1.5 times, while the expression of miR-21 remained unchanged (Fig. 3A). These results suggest that the targeted degradation of Lin28A by P7f-1-FL-P4-C specifically led to the enhanced expression levels of all mature Let-7 family miRNAs. The up-regulation of mature miRNA Let-7 expression also followed a dose- and time-dependent pattern. Increasing the concentration of P7f-1-FL-P4-C from 25 to 200 nM, the let-7g expression level gradually increased and reached the plateau at 100 nM. With incubation time increase after P7f-1-FL-P4-C (100 nM) treatment, the let-7g level also gradually increased and reached 1.8-fold at 72 hours after transfection (Fig. 3, B and C). P7f-1-FL-ORN and P7f-1-FL-ORN physically mixed with pomalidomide could only increase the let-7g level to about 1.2-fold, and pomalidomide itself could not increase the let-7g level (Fig. 3D). This finding is consistent with the results of the Lin28A degradation, suggesting that the up-regulation of mature miRNA let-7 expression following Lin28A degradation mediated by P7f-1-FL-P4-C is due to a specific on-target effect of Lin28A-miRNA-PROTAC, rather than a nonspecific effect of any individual component. The results of miRNA omics analysis further proved that the treatment of P7f-1-FL-P4-C could substantially increase the expression of let-7 miRNA family (fig. S8).

Fig. 3. The expression of mature miRNA let-7.

(A) The degradation of Lin28A by P7f-1-FL-P4-C resulted in alterations in the expression level of mature miRNA let-7 in T47D cells. (B) Concentration effect of P7f-1-FL-P4-C on the up-regulation of let-7g expression following the degradation of Lin28A. (C) Time effect of P7f-1-FL-P4-C on the up-regulation of let-7g expression following the degradation of Lin28A. (D) Control experiment was conducted to assess the expression of let-7g. Values are means ± SEM with n = 3. P values were calculated with one-way ANOVA test. *P < 0.05; **P < 0.01; ****P < 0.0001.

Antitumor activity of Lin28A-miRNA-PROTAC in vitro

The inhibitory effects of miRNA PROTAC molecule P7f-1-FL-P4-C on the proliferation of T47D were assessed. Notably, only P7f-1-FL-P4-C treatment substantially inhibited T47D cell proliferation and more cell toxicity was observed with extended exposure of P7f-1-FL-P4-C (Fig. 4A), which was superior to that observed with only P7f-1-FL-ORN treatment. Meanwhile, when compared to P7f-1-FL-ORN, treatment with P7f-1-FL-P4-C markedly suppressed clonal formation and migration of T47D cells, resulting in a one-third reduction in colony number and one-half decrease in cell migration ability (Fig. 4, C and E, and fig. S9). Moreover, a 1.75-fold increase in cell apoptosis and cell cycle arrest was induced (Fig. 4D and fig. S10). Further, by applying a mutated P7f-1-FL-P4-C, we could observe that the loss of binding affinity between mutated P7f-1-FL-P4-C and Lin28A led to the low inhibitory effect of T47D cell proliferation (fig. S11).

Fig. 4. Lin28A-miRNA-PROTAC and TAM synergistically inhibit T47D cell proliferation and migration.

(A) The proliferation of T47D cells was suppressed by P7f-1-FL-P4-C. (B) The cell viability of T47D cells was assessed following treatment with P7f-1-FL-P4-C alone, TAM alone, or a combination of P7f-1-FL-P4-C and TAM. (C) The colony formation of T47D cells is inhibited by P7f-1-FL-P4-C alone or in combination with TAM. (D) The combination of P7f-1-FL-P4-C with TAM or its individual administration leads to an increase in apoptosis of T47D cells. (E) The migration of T47D cells is inhibited by P7f-1-FL-P4-C alone or in combination with TAM. Values are means ± SEM with n = 3. P values were calculated with one-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

TAM, a drug that suppresses estrogen receptors, was found to develop resistance in 50% of breast cancer patients as noted in clinical studies (43). To verify the sensitivity of chemotherapeutics to cancer cells induced by P7f-1-FL-P4-C PROTAC, T47D cells were treated with P7f-1-FL-P4-C and TAM either individually or in combination. Treatment with TAM at 25 μM induced the similar cell toxicity and cell proliferation as that of 100 nM P7f-1-FL-P4-C treatment. However, the combined treatment of P7f-1-FL-P4-C with TAM exhibited much higher cytotoxicity than either treatment alone. The synergistic effect was most pronounced when 100 nM P7f-1-FL-P4-C and 25 μM TAM were used (Fig. 4B). In comparison to individual treatment, the combined approach of P7f-1-FL-P4-C and TAM substantially suppressed the clonal formation with around sixfold reduction and enhanced cell apoptosis by twofold (Fig. 4, C and D). In addition, almost no migration of T47D cells was observed with P7f-1-FL-P4-C and TAM combined treatment (Fig. 4E and fig. S3).

Antitumor activity of Lin28A-miRNA-PROTAC in vivo

With encouraging cellular results, the therapeutic efficacy of P7f-1-FL-P4-C and TAM in T47D allogeneic transplant mice was evaluated. P7f-1-FL-P4-C (3 mg/kg) was encapsulated in a mix of neutral cytidine lipid DNCA(2-(4-amino-2-oxopyrimidin-1-yl)-N-(2,3-dioleoyl-oxypropyl) acetamide)/cationic lipid CLD (dioleoyl-3,3′-disulfanediylbis-[2-(2,6-diaminohexanamido)] propanoate)/DSPE-PEG2000 (44) and administered via intratumoral injection. TAM (5 mg/kg) in corn oil was administered by gavage. Similarly, P7f-1-FL-P4-C and TAM were also administered individually for comparison every 3 days (Fig. 5A). Throughout the treatment, no notable variations were observed in the body weight of mice or the weight of the normal tissues (Fig. 5B and fig. S4), indicating no obvious toxicity of P7f-1-FL-P4-C and TAM when administered either individually or in combination. After P7f-1-FL-P4-C and TAM treatment, tumor volume and weight were recorded, revealing that only P7f-1-FL-P4-C PROTAC treatment effectively inhibited tumor growth, demonstrating a 2.5-fold decrease in tumor size and a 50% reduction in tumor weight compared to P7f-1-FL-ORN treatment (Fig. 5, C to E). In addition, the combined administration of P7f-1-FL-P4-C and TAM substantially inhibited tumor growth, with the observable tumor regression after 2 weeks (Fig. 5, C to E), resulting in a 7.5- and 2.5-fold decrease compared to negative control and P7f-1-FL-P4-C or TAM treatment groups. The tumor weights were also reduced by seven- and threefold in comparison to control and P7f-1-FL-P4-C or TAM treatment groups.

Fig. 5. Lin28A-miRNA-PROTAC and TAM combination therapy inhibits tumor growth.

(A) Mode and frequency of administration of mice. (B) Alterations in the body weight of mice during the course of the treatment (n = 5 mice per group). (C) Tumor growth curves of the groups of mice following the specified treatments. Values are means ± SEM with n = 5. P values were calculated with one-way ANOVA test. ***P < 0.001; ****P < 0.0001. (D) Photos of T47D allograft tumors in groups of mice following indicated treatments (n = 5 mice per group). (E) Tumor weight of mouse groups after indicated treatments. Values are means ± SD with n = 5. P values were calculated with one-way ANOVA test. ***P < 0.001; ****P < 0.0001. (F) Lin28A protein levels of T47D allograft tumors from mouse groups after indicated treatments. Values are means ± SEM with n = 3. P values were calculated with one-way ANOVA test. **P < 0.01; ***P < 0.001. (G) Representative images of H&E (scale bar, 100 nm) and TUNEL (scale bar, 50 nm) and IHC (scale bar, 100 nm) staining for Lin28A in T47D tumor tissue after indicated treatments.

Following tumor section, Lin28A protein levels in tumor tissues of different groups were also assayed, revealing that Lin28A levels in tumors from mice treated with P7f-1-FL-P4-C alone or its combination with TAM were substantially reduced to around 20 to 30% of the control. However, TAM treatment alone did inhibit tumor growth, but it did not obviously reduce the Lin28A levels (Fig. 5F), demonstrating the effective in vivo degradation of Lin28A protein by P7f-1-FL-P4-C. Hematoxylin and eosin (H&E) staining also confirmed that the cancer cell density of tumor tissues with individual P7f-1-FL-P4-C–treated mice already greatly decreased compared to that of the control group, while the combined administration group exhibited much lower cancer cell tissue density than the single administration group with P7f-1-FL-P4-C or TAM individually (Fig. 5G). According to the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining results, clear apoptotic signals were observed in the single administration group P7f-1-FL-P4-C or TAM, and a large area of cancer cell apoptosis was successfully achieved in the combined administration group, exhibiting large vacuoles and more intense dead cells in tumor tissues compared to the group treated with P7f-1-FL-P4-C or TAM individually (Fig. 5G). Pre-let-7f-1 (P7f-1-FL-ORN) oligonucleotide itself did not have beneficial effect on tumor inhibition in our in vivo experiments (Fig. 5), suggesting that the introduction of pre-let-7 miRNA alone might not be a suitable choice for tumor therapy. Immunohistochemistry (IHC) staining of Lin28A protein demonstrated that P7f-1-FL-P4-C, either alone or in combination with TAM, effectively reduced Lin28A protein levels in tumor tissues compared to the negative control (NC) group and P7f-1-FL-ORN group (Fig. 5G). All these results confirmed that P7f-1-FL-P4-C PROTAC mediates the degradation of the endogenous protein Lin28A in tumors and has a synergistic effect with TAM, exerting a potent antitumor effect.

DISCUSSION

Various miRNA-based therapies have been developed due to the fact that miRNA plays an important role in the pathogenesis of numerous diseases. However, none of them currently progressed to phase III clinical trials. The biogenesis of miRNA is a multifaceted process that encompasses various stages, starting from nuclear transcription to the production of mature miRNA in the cytoplasm (45). Numerous RBPs play a crucial role in regulating the biogenesis and maturation of miRNAs. They primarily influence miRNA processing by either promoting or inhibiting it through their interactions with canonical proteins, such as DROSHA and DICER (46). The expression levels of RBPs have been found to be closely associated with the level of mature miRNAs. Therefore, achieving a sustainably high level of therapeutic endogenous tumor suppressor miRNAs can be realized by degrading certain RBPs that inhibit the maturation of therapeutic miRNAs, offering the promise for miRNA-based therapy.

The pre-let-7 sequence, exhibiting a strong affinity for RBP Lin28A, was chosen as the foundation for developing miRNA-based PROTACs for cancer therapy. A series of miRNA-based PROTACs derived from pre-let-7f-1 sequence were synthesized and characterized. Lin28A-miRNA-PROTACs with full-length pre-let-7f-1 oligonucleotide showed highly effective Lin28A degradation under the same conditions through a ubiquitination process. Lin28A-miRNA-PROTACs with only CSD binding oligonucleotide sequences still maintain Lin28A degradation in some Lin28A-miRNA-PROTAC instances. Lin28A-miRNA-PROTACs with only ZKD targeting oligonucleotide sequence lost the ability of inducing Lin28A degradation. Among these Lin28A-miRNA-PROTACs, P7f-1-FL-P4-C with PEG linker and CRBN ligand could specifically reduce the expression level of Lin28A down to 20% at a low concentration (50 nM) in a short time (12 hours, or even less) through a ubiquitin-proteasome–dependent manner. Subsequently, it promoted the up-regulation of mature let-7 family miRNAs, exhibiting a tumor-suppressing effect in T47D cells and resulting in the inhibition of T47D cell proliferation and migration. Further combination of P7f-1-FL-P4-C and TAM markedly suppressed the proliferation, migration, and clonal formation of T47D cells, as well as the induced cell apoptosis and cell cycle arrest. In vivo mice tumor models demonstrated that P7f-1-FL-P4-C effectively degraded Lin28A and inhibited the growth of T47D tumors. The combined administration of P7f-1-FL-P4-C with TAM resulted in a more pronounced growth inhibition of T47D tumors, with a tendency toward tumor regression. Meanwhile, whether administered individually or in combination, there is no obvious toxicity to mice under the treatment conditions. This study offers a promising therapeutic approach for treating breast cancer and also provides an effective miRNA-based PROTAC strategy to facilitate miRNA therapy.

Similar to the most nucleic acid drugs, this type of PROTACs has a high molecular weight and necessitates an appropriate delivery strategy to ensure effective administration. Here, DNCA/CLD/PEG was used for intratumoral delivery to assess the impact of PROTAC P7f-1-FL-P4-C. Novel nucleic acid delivery technologies with organ-specific targeting methods have recently been developed, enabling the systematic application of miRNA-based PROTACs. At the same time, natural nucleotides are susceptible to degradation by intracellular nucleases, making nucleotide modification essential. Here, total thiophosphate was used, and more modification methods may bring better degradation activity, which needs more exploration in the future.

MATERIALS AND METHODS

Chemical synthesis

The reagents and solvents used in this study were bought from Tongguang, Solarbio, OU HE, Energy Chemical, Sigma-Aldrich, TCI, and 3AChem. All reagents and solvents for organic synthesis were purchased from commercial suppliers without further purification. Nuclear magnetic resonance spectra were obtained on a Bruker Advance III 400 spectrometer. Electrospray ionization mass spectra were measured on a Waters SQD mass spectrometer. Detailed synthesis methods can be found in the Supplementary Materials.

Synthesis of oligonucleotides and Lin28A-miRNA-PROTAC

All RNA oligonucleotides were synthesized on an LK-48E DNA/RNA synthesizer based on standard phosphoramidite solid-phase synthesis. RNA oligonucleotides were synthesized based on universal CPG. 3′-C7 NH2 RNA oligonucleotides were synthesized based on 3′-Amino-Modifier C7 CPG (GenePharma, China). 3′-C7 NH2 RNA oligonucleotides (10.0 nmol, 1.0 equiv) were dissolved in 50 μl of 1× phosphate-buffered saline (PBS), and NHS reactive esters (5.0 μmol, 500 equiv) of CRBN/VHL ligands of different lengths synthesized above were dissolved in 50 μl of dimethyl sulfoxide (DMSO), and then the two components were mixed and shaken for 12 hours at room temperature. Lin28A-miRNA-PROTAC was purified by HPLC after dialysis in a 1000-dalton dialysis tubing.

Cell culture and transfection

The T47D cell line was purchased from Procell (human, CL-0228). T47D cells were cultured in RPMI 1640 [containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin and insulin (10 μg/ml)]. Lin28A-miRNA-PROTAC was transfected into cells using Lipofectamine 2000 according to the standard protocol instructions.

Stability experiment of pre-let-7f-1 with cell lysate

T47D cells (2 × 106) were gathered to a centrifuge tube and washed with PBS solution, followed by lysis in 0.5 ml of lysis buffer containing 10 mM Pipes-NaOH (pH 6.8), 300 mM sucrose, 100 mM NaCl, 1 mM EGTA, 3 mM MgCl2, and 0.5% Triton X-100. Subsequently, the residues were centrifuged at 14,000g for 15 min at 4°C, and liquid supernatants were collected. The 6-FAM–labeled pre-let-7 miRNAs (20 pmol, 1 μl) were added into the cell lysate (9 μl) and incubated at 37°C. The samples were removed after the indicated amounts of time and immediately frozen in liquid nitrogen. Following the addition of 2× RNA loading buffer, the aliquots were analyzed on 20% polyacrylamide gels at 250 V for 40 min.

Western blot

Cells were lysed in radioimmunoprecipitation assay lysate, and protease inhibitor cocktail (100×) was added. Protein concentration was measured with the BCA Protein Quantification Kit. The lysate (20 μg of protein) was electrophoresed with 10% SDS-PAGE (polyacrylamide gel electrophoresis) at 120 V for 1 hour. Protein (30 μg) of each sample was loaded on an SDS-PAGE gel, electrophoresed, and transferred to a nitrocellulose filter membrane of 0.45-μm pore size (Millipore). The membranes were blocked with 5% nonfat milk in tris-buffered saline with 0.1% Tween 20 (TBST) and incubated with the indicated antibody overnight at 4°C. After washing with TBS buffer containing 0.1% Tween 20 for four times, the nitrocellulose filter membrane was incubated with secondary antibody at room temperature for 1 hour, followed by another four washes with TBS buffer for post-imaging. Anti-Lin28A antibody was purchased from Abcam (ab124765, 1:2000), and anti–α-tubulin antibody was purchased from EASYBIO (1:5000). Goat anti-rabbit immunoglobulin G (IgG) secondary antibody (DyLight 800, A23920, 1:10,000) and goat anti-mouse IgG secondary antibody (DyLight 800, A23910,1:10,000) were purchased from Abbkine. All primary antibodies were diluted with a dilution of the primary antibody. Secondary antibodies are diluted with 5% skim milk in TBST buffer solution.

Lin28A mRNA RT-PCR

Cells were collected, and total RNA was extracted from cells with Biozol reagent (Vazyme). Then, 300 ng of RNA was reverse transcribed to cDNA with HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was run with GoTaq qPCR master mix (Promega) using an ABI QuantStudio 6. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers used were listed as follows: Lin28A: 5′-AAGCGCAGATCAAAAGGAGA-3′ (forward) and 5′-CTGATGCTCTGGCAGAAGTG-3′ (reverse); GAPDH, 5′-ATGTTCGTCATGGGTGTGAA-3′ (forward) and 5′-GGTGCTAAGCAGTTGGTGGT-3′ (reverse).

Coimmunoprecipitation

Lin28A antibody (ab124765) was cross-linked to Dynabeads protein A (Thermo Fisher Scientific, 10001D) according to the manufacturer’s protocol. Cell lysates were incubated with Lin28A-Dynabeads overnight at 4°C. Lin28A-immunoprecipitated complexes were washed three times with PBS. Proteins were eluted by boiling in loading buffer and then processed for Western blot analysis. CRBN antibody was bought from Abcam (ab315344). Normal rabbit IgG was bought from Cell Signaling Technology (2729S). HRP, Mouse Anti-Rabbit IgG LCS was bought from Abbkine (A25022).

In vitro ubiquitination assay

Briefly, T47D cells were first transfected with His-ub plasmid for 24 hours, followed by additional transfection of P7f-1-FL-P4-C for another 24 hours. Cells were treated with 20 μM MG132 for 4 hours before harvesting cells. After sonication with denaturing lysate B (8 M urea, 100 mM NaH2PO4, 10 mM tris, 25 mM imidazole, pH 8.0), the cell lysate was incubated with Ni-IDA-Sefinose Resin for 4 hours at 4°C. The Ni-IDA-Sefinose Resin was washed three times in buffer solution C (8 M urea, 100 mM NaH2PO4, 10 mM tris, 25 mM imidazole, pH 6.3) and twice in buffer E (8 M urea, 100 mM NaH2PO4, 10 mM tris, 250 mM imidazole, pH 4), and finally, the ubiquitinated protein was separated with 10% SDS-PAGE and immunoblotted with individual antibody.

CRBN knockdown

CRBN targeting siRNA (siCRBN) was designed with sense strand (5′-CCCAGACACUGAAGAUGAAAU-3′) and antisense strand (5′-AUUUCAUCUUCAGUGUCUGGG-3′). The annealed siCRBN was transfected according to Lipofectamine 2000 by the manufacturer’s manual. After 24 hours, the cells were further transfected with P7f-1-FL-P4-C and continued to culture for 48 hours.

MS analysis and miRNA sequencing analysis

For proteomic analysis and miRNA sequencing analysis, T47D cells were inoculated in 100-mm petri dishes at a density of 3 × 106 per dish (n = 3) and incubated for 24 hours. P7f-1-FL-P4-C and pre-let-7f-1 were transfected into cells using Lipofectamine 2000 at a concentration of 100 nM, and the culture was continued for 48 hours. The cells were collected for MS analysis and miRNA sequencing analysis. Cells grown in complete medium for the same time were used as controls (n = 3). miRNA sequencing analysis was performed by GENEWIZ Company, and MS analysis was performed by Peking University Analytical Testing Platform.

miRNA RT-PCR

Cells were collected and treated with trypsin, and total RNA was collected. The miRcute miRNA isolation kit, miRcute miRNA cDNA synthesis kit, and miRcute Plus miRNA qPCR kit (Tiangen Biotechnology, Beijing) were used to extract RNA from cells, perform reverse transcription reaction, and then perform real-time PCR detection of miRNA, according to the standard protocol provided by the manufacturer. The threshold period for each sample was normalized to U6 miRNA.

Cell proliferation

T47D cells were seeded in 96-well plates at a density of 1.5 × 104 cells per well. After 12 hours of culture, P7f-1-FL-ORN and P7f-1-FL-P4-C were transfected with Lipofectamine 2000 at a concentration of 100 nM for an appropriate duration. In the P7f-1-FL-P4-C group in combination treatment, T47D cells were seeded in 12-well plates at a density of 2 × 105 cells per well. After 12 hours of culture, P7f-1-FL-P4-C was transfected with Lipofectamine 2000 at a concentration of 100 nM. Following a 12-hour incubation period, T47D cells were reseeded in 96-well plates at a density of 1.5 × 104 cells per well for a suitable duration. For both the TAM group and the coadministration group, the cells were exposed to varying concentrations of TAM (5, 10, and 25 μM) for 48 hours. Subsequently, 10 μl of MTT solution (5 mg/ml, prepared in PBS, pH 7.4) was added to each well, and the cells were further incubated for 4 hours. Following this, 150 μl of DMSO was added to each well, and the absorbance at 490 nm was measured using a microplate reader to calculate cell viability.

Clonal formation

A total of 5 × 105 cells were seeded in each well of a six-well plate. P7f-1-FL-ORN and P7f-1-FL-P4-C were transfected with Lipofectamine 2000 at a concentration of 100 nM. Following a 12-hour transfection period, the cells in each well were enumerated, and subsequently, 1000 cells per well were seeded in a six-well plate. After 12 hours, the TAM group was supplemented with a final concentration of 25 μM TAM and cultured for an additional 2 weeks. The cells were immobilized using paraformaldehyde and stained with 0.1% crystal violet.

Apoptosis assays

A total of 2 × 105 cells were plated in each well of 12-well plates. P7f-1-FL-ORN and P7f-1-FL-P4-C were transfected with Lipofectamine 2000 at a concentration of 100 nM. The TAM group and the combination group were treated with a final concentration of 25 μM TAM and incubated for 48 hours. Subsequently, the cells were harvested, centrifuged, washed with PBS, and then gently resuspended in 195 μl of binding buffer, 5 μl of annexin V–fluorescein isothiocyanate (FITC), and 10 μl of propidium iodide staining solution. The samples should be incubated at room temperature in the absence of light for a duration of 20 min, and were subsequently analyzed using flow cytometry.

Cell cycle assays

A total of 2 × 105 cells were plated in each well of 12-well plates. P7f-1-FL-ORN and P7f-1-FL-P4-C were transfected with Lipofectamine 2000 at a concentration of 100 nM. The TAM group and the combination group were treated with a final concentration of 25 μM TAM and incubated for 48 hours. Subsequently, the cells were harvested, centrifuged, washed with prechilled PBS, and then fixed with prechilled 70% ethanol overnight at 4°C. The samples were treated with propidium iodide staining solution, incubated at 37°C for 30 min in the absence of light, and were subsequently analyzed using flow cytometry.

Scratch assays

Cells (5 × 105) were seeded in each well of a six-well plate. P7f-1-FL-ORN and P7f-1-FL-P4-C were transfected with Lipofectamine 2000 at a concentration of 100 nM. Following a 12-hour transfection period, the cells in each well were enumerated. The Culture-Insert was placed in a six-well plate, and 5 × 104 cells (100 μl) were added to each of the Culture-Insert 2 wells. The cells were then incubated for 24 hours. The Culture-Inserts were carefully extracted using sterile forceps, followed by washing the cell layer with PBS and addition of 2 ml of fresh medium (TAM-containing medium was added to the TAM group and the combination group). Cell images were captured at 0, 12, 24, and 48 hours using an automated inverted fluorescence microscope, and the findings were subsequently analyzed by ImageJ.

Tumor growth inhibition

All animal experiments were approved by the Committee for Animal Research of Peking University (permission certificate no. SYXK 2022-0037). Six-week-old BALB/c male nude mice (Department of Laboratory Animal Science of the Peking University Health Science, Beijing, China) were used to establish the T47D mice model. All cells were injected in a 100-μl volume of growth medium containing 50% Matrigel. To stimulate tumor growth, T47D mice were subcutaneously injected in the scapular region with 0.1 ml of β-estradiol 17-valerate (5 mg/ml in sesame oil). Upon reaching a tumor volume of approximately 50 mm3, T47D mice were randomly allocated into five groups. The initial group received an intratumoral injection of 0.9% normal saline. The second group received intratumoral injections of P7f-1-FL-ORN at a dosage of 6 nmol per piece. The third group received intratumoral injections of P7f-1-FL-P4-C at a dosage of 6 nmol per piece. The fourth group received TAM (5 mg/kg, dissolved in corn oil) via gavage, while the fifth group was subjected to intratumoral injection of P7f-1-FL-P4-C (6 nmol per piece) and intragastric administration of TAM (5 mg/kg). Upon completion of the treatment, the major diameter (a) and minor diameter (b) of the tumors were measured, and the tumor volume was computed using the formula 1/2 × a × b2. Following the treatment, the tumors were excised and their weights were recorded. Part of the tumor tissues were submerged in 4% paraformaldehyde at 4°C and subsequently analyzed using H&E, TUNEL, and IHC. Lin28A antibody for IHC was bought from Abcam (ab124765). The remaining portions of the same tumor tissues were preserved in liquid nitrogen for a Western blot assay.

Statistical analysis

Data are represented as means ± SEM. For statistical analysis between two groups, two-tailed Student’s t test was applied. For multiple comparison, one-way analysis of variance (ANOVA) was applied. Statistically significant differences were expressed as follows: “ns” represents P > 0.05, “*” represents P < 0.05, “**” represents P < 0.01, and “***” represents P < 0.0001. Statistical analysis was performed with GraphPad Prism software (version 8.0.1).

Acknowledgments

Funding: This work was supported by the National Natural Science Foundation of China (grant nos. 22277003, 81821004, 22077005, 22107004, and 22077008) and the National Key R&D Program of China (2022YFC2303700).

Author contributions: Conceptualization: X.T. Investigation: J.X., X.Z., X.L., and D.G. Methodology: J.X., X.Z., Q.W., and X.T. Data curation: X.T. Validation: J.X., X.Z., X.L., D.G., and Q.W. Formal analysis: J.X., X.Z., X.L., and D.G. Resources: Q.W. Visualization: J.X. and X.Z. Supervision: X.T. Writing—original draft: J.X., X.T., and X.Z. Writing—review and editing: J.W., Q.W., and X.T. Funding acquisition: J.W., Q.W., and X.T. Project administration: X.T.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The compounds and molecules prepared in this study can be provided by the authors pending scientific review and a completed material transfer agreement.

Supplementary Materials

This PDF file includes:

Figs. S1 to S12

Tables S1 to S4

Synthetic Procedures and Characterization
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