A 3,4-diamino-substituted coumarin derivative as a TRBP inhibitor suppresses tumor growth in breast cancer by inducing cellular senescence

Abstract

Transactivation response RNA-binding protein 2 (TRBP) mediates microRNA (miRNA) biogenesis and regulates messenger RNA (mRNA) stability. It is a pivotal regulator of post-transcriptional gene expression, influencing processes including cellular senescence and tumorigenesis. Elevated TRBP expression correlates with poor prognosis in breast cancer, underscoring its potential as a therapeutic target. We identified a 3,4-diamino-substituted coumarin derivative (3ai), a novel small-molecule compound inhibitor of miR-21 biogenesis. Compound 3ai increased the expression of tumor-suppressor proteins targeted by miR-21. Further mechanistic studies revealed that 3ai binds TRBP and disrupts the biogenesis of senescence-associated miRNAs. This interaction induces cell cycle arrest and DNA damage in tumor cells, ultimately promoting cellular senescence in breast cancer cells and suppressing their proliferation and metastasis potential. Our study reveals that 3ai directly engages TRBP to modulate miRNA biogenesis, thereby inducing cellular senescence. These results support TRBP as a therapeutic target in breast cancer and warrant further development of 3ai as a candidate therapeutic for breast cancer.

Introduction

Breast cancer exhibits pronounced intratumoral heterogeneity and high global prevalence. Genomic instability, in concert with a dynamic tumor microenvironment, drives therapeutic resistance and constrains clinical efficacy [1]. Although conventional chemotherapy remains a cornerstone of management, its benefit is limited by dose-dependent toxicities, acquired resistance, and poor tumor selectivity. Cellular senescence, defined as a stable proliferative arrest, is accompanied by extensive transcriptional reprogramming, increased protein synthesis, and acquisition of a senescence-associated secretory phenotype (SASP) [2]. In terms of its anti-tumor effect [3], cellular senescence enforces persistent cell cycle arrest suppresses tumor initiation. Through the SASP, senescent cells can propagate senescence to adjacent tumor cells, creating a barrier that impedes tumor growth [4]. Therapeutic strategies targeting tumor cellular senescence via stable cell cycle arrest are increasingly recognized as viable in breast cancer, with translational promise owing to their dual capacity to suppress proliferation and enhance treatment responsiveness [5]. For example, deuterium-depleted water (DDW) combined with cisplatin has been reported to promote senescence in triple-negative breast cancer (TNBC) MDA-MB-231 cells [6]. Likewise, inducting tumor-cell senescence and apoptosis has been demonstrated to effectively suppress TNBC and HER2-positive breast cancer while reducing recurrence risk [7]. Collectively, these findings underscore the clinical potential of targeting tumor senescence. Accordingly, identifying therapeutic targets and agents that induce tumor-cell senescence may open new avenues for breast cancer treatment.

MicroRNAs (miRNAs) are small non-coding RNA molecules that act as post-transcriptional modulators of gene expression via RNA-induced silencing complex (RISC), leading to translational repression or mRNA degradation [8]. The complex gene networks regulated by miRNAs influence not only tumor cell proliferation and metastasis but also the expression of senescence-associated genes, thereby promoting cellular senescence [9,10]. Several senescence-associated miRNAs-including miR-21, miR-34a, and miR-146 are aberrantly expressed in breast cancer and contribute to tumor progression and metastasis [11-14]. In addition, specific miRNAs have been shown to inhibit tumor growth by promoting cellular senescence [15,16]. Previous studies have shown that the upregulation of let-7d and miR-335 could inhibit cellular senescence [17,18]. Notably, miR-21 levels in serum exosomes are significantly higher in breast cancer patients with bone metastases than in those without [19]. MiR-21 functions as an oncogenic miRNA, and its overexpression is strongly associated with breast cancer progression [20]. Modulating the expression of these miRNAs may help control breast cancer progression. Transactivation response RNA-binding protein 2 (TRBP) is an RNA-binding protein that plays a critical role in miRNA biogenesis [21,22]. As a component of the RISC-loading complex, TRBP assists Dicer and Argonaute 2 (AGO2) in pre-miRNA processing and strand selection and loading, thereby ensuring accurate miRNA maturation. TRBP is required for efficient miRNA production and target mRNA repression, thereby broadly influencing gene expression [23]. TRBP has emerged as a therapeutic target in cancer [22]. In breast cancer, TRBP promotes invasion and metastasis, and high tumor expression is significantly associated with poor patient survival [24]. Therefore, identifying agents that induce tumor-cell senescence by targeting TRBP to modulate miRNA biogenesis may offer new therapeutic opportunities for breast cancer.

In this study, we used a previously established HeLa-miR-21-luciferase reporter cell line [25] to conduct a high-throughput screen of 202 synthetic and natural compounds. We identified a small molecule, 3,4-diamino-substituted dihydrocoumarin (3ai) [26], that selectively inhibits TRBP, thereby disrupting miRNA biogenesis. In breast cancer models, 3ai induced tumor-cell senescence, indicating therapeutic potential.

Materials and methods

Cell line and cell culture

The HeLa-miR-21-luciferase stable cell line was obtained from the Chengdu Institute of Biology, Chinese Academy of Sciences, Sichuan, China. The HeLa-miR-21-luciferase cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; VivaCell Bioscience, C3103). The human breast cancer cell lines MDA-MB-231 and MCF-7, along with the non-tumorigenic breast epithelial cell line MCF-10A, were obtained from Procell, Life Science & Technology Co., Ltd., Wuhan, China. The MDA-MB-231 cell line was maintained in DMEM, whereas MCF-7 cells were cultured in Minimum Essential Medium (MEM, Gibco, 612053) supplemented with 1% glutamine (Beyotime, C0212), 1% non-essential amino acids (100×; Beyotime, C0332), 1% sodium pyruvate (100 mM; Beyotime, C0331), and 0.01 mg/mL recombinant human insulin (Procell, PB180432). Both media were supplemented with 10% (v/v) FBS (VivaCell Bioscience, C04001) and 1% penicillin-streptomycin (VivaCell Bioscience, C3420). MCF-10A cells were cultured in specific growth medium (Procell, CM-0525). All cells underwent STR authentication.

Luciferase activity assay

HeLa-miR-21-luciferase cells were seeded into 96-well plates at a density of 1 × 10⁵ cells per well and incubated overnight in a 5% CO₂ atmosphere. The following day, cells were treated with DMSO (vehicle control) or various concentrations of 3ai for 24 h. Cell lysates were then prepared using an optimized passive lysis buffer and incubated under controlled thermal conditions (22 ± 0.5°C) for 15 min. Luminescence intensity was quantified with a multi-mode microplate reader (Synergy^TM H1; BioTek, Agilent Technologies).

Quantitative Real-Time PCR (qRT-PCR)

Total RNA was isolated from treated cells using TransZol Up reagent (TransGen Biotech, ET111-01-V2, Beijing, China). Cell lysates were homogenized and incubated with 1/5 volume of RNA extraction reagent, followed by centrifugation at 15,000×g for 15 min at 4°C. The supernatant was transferred to a new tube and mixed gently with an equal volume of isopropanol at RT for 10 min for RNA precipitation. After another centrifugation at 15,000×g for 15 min at 4°C, the RNA pellet was washed twice with 75% ethanol and air-dried for 5 min, then dissolved in 20-50 μL RNA dissolving solution. Total RNA was reverse transcribed into cDNA using the miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, MR101-02) along with Bulge-Loop^TM miRNA-specific primers, following the manufacturer’s instructions. The synthesized cDNA was stored at -20°C for subsequent use. Quantitative PCR was performed using TransGen Biotech’s PerfectStart^® Green qPCR SuperMix (AQ602). The relative gene expression levels were calculated using the 2^-ΔΔCt method, with U6 small nuclear RNA serving as the internal control. Stem-loop structured reverse transcription primers were designed for microRNA detection, and the corresponding sequences are listed in Table S1. All reactions were performed in triplicate, and no-template controls were included to ensure amplification specificity. Total RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase and Bulge-Loop^TM miRNA-specific primers in a two-step protocol: 42°C for 15 min (reverse transcription) followed by 85°C for 5 s (enzyme inactivation). The synthesized cDNA was stored at -20°C until further use. For quantitative real-time PCR (qRT-PCR), reactions were performed on a LightCycler 480 system (Roche) with U6 small nuclear RNA as the internal control. MicroRNA reverse transcription primers were designed using the stem-loop method, and the corresponding sequences are provided in Table S1. All reactions were conducted in triplicate, and no-template controls were included to verify assay specificity.

Western blotting

Cells were lysed in RIPA buffer (Beyotime, P0013C) supplemented with protease and phosphatase inhibitors. Following centrifugation, protein concentrations were determined using a BCA protein assay kit (Beyotime, P0009). Equal amounts of protein were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and blocked with 5% bovine serum albumin (BSA). The membranes were incubated overnight at 4°C with the appropriate primary antibodies, followed by incubation with secondary antibodies for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) substrate (Proteintech, PK10003), and band intensities were quantified using ImageJ software. Relative protein expression levels were normalized to GAPDH.

Cellular Thermal Shift Assay (CETSA)

Cells were harvested, resuspended in phosphate-buffered saline (PBS) supplemented with 10% (v/v) protease inhibitor cocktail, and subjected to three freeze-thaw cycles in liquid nitrogen. For each cycle, the suspension was frozen in liquid nitrogen for 15 s and then thawed in a 37°C water bath for 5 min, repeated three times. Cell lysates were obtained by centrifugation of the homogenate at 20,000 g for 20 min at 4°C to remove cellular debris. The resulting supernatant was aliquoted according to predefined concentrations or temperature conditions of compound 3ai and vehicle controls. Lysates were incubated at 37°C for 30 min and subsequently divided into 50 μL aliquots. These portions were subjected to thermal denaturation by incubating them at the specified temperatures for 3 min, followed by rapid cooling on ice for 3 min to halt the thermal treatment. To separate soluble proteins from insoluble aggregates, the lysates were centrifuged again at 20,000 g for 20 min at 4°C. The supernatants were transferred to new microcentrifuge tubes, avoiding precipitate contamination, and analyzed by SDS-PAGE.

Surface Plasmon Resonance (SPR) assay

Surface plasmon resonance (SPR) analysis was performed on a BIAcore T200 system (Cytiva) according to established protocols [25]. A CM5 sensor chip was functionalized by sequential injection of a 1:1 mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N’-(dimethylaminopropyl) carbodiimide (EDC) to enable covalent immobilization. Recombinant human TRBP protein (20 μg/mL in 10 mM sodium acetate buffer, pH 4.5) was immobilized onto the activated surface via amine coupling, followed by deactivation of residual reactive groups with 1 M ethanolamine hydrochloride (pH 8.5). A reference flow cell subjected to activation and blocking without protein immobilization was used for background subtraction. Serial dilutions of compound 3ai prepared in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20, pH 7.4, supplemented with 1% DMSO) were injected across the chip surface at a flow rate of 10 μL/min for 420 s, followed by a dissociation phase of 300 s. Baseline regeneration between analyte cycles was performed with 50 mM NaOH to restore resonance signals. Sensorgram data were analyzed with BIAevaluation 3.2 software (Cytiva), and equilibrium dissociation constants (KD) were calculated using steady-state affinity models to quantify TRBP-3ai interactions.

Assays for cell proliferation and viability

To evaluate the effect of 3ai on cell viability, MCF-10A, MCF-7, and MDA-MB-231 cells were seeded into 96-well plates at a density of 5 × 10⁴ cells and allowed to adhere overnight. The cells were treated with 0.1% DMSO (control) or various concentrations of 3ai. After 24 h, 10% (v/v) Cell Counting Kit-8 (CCK-8; Beyotime, C0039) solution was added to each well, and the cells were incubated for 30-60 min. Absorbance at 450 nm was measured using a multi-mode microplate reader (BioTek Synergy^TM H1, Agilent Technologies).

Colony formation assay

MCF-7 and MDA-MB-231 cells were seeded into 6-well plates at a density of 4000 cells per well, allowed to adhere overnight, and then treated with different concentrations of 3ai or 0.1% DMSO (vehicle control). After a 7-14 day incubation period, colonies were fixed with 4% paraformaldehyde for 15 min. The fixed colonies were stained with 0.1% crystal violet solution (Solarbio, C8470), rinsed with double-distilled water, air-dried at room temperature, and subsequently imaged using a digital camera.

Transwell migration assay

MCF-7 and MDA-MB-231 cells were seeded in the upper chamber at a density of 1 × 10⁵ cells per well in 200 μL serum-free MEM. The lower chamber was filled with 600 μL of complete MEM supplemented with 10% FBS. After treatment with 3ai, the cells were allowed to migrate for 12-16 h at 37°C. Non-migrated cells in the upper chamber were gently removed with a cotton swab, and the migrated cells on the lower membrane surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Images were acquired using a light microscope.

Immunofluorescence

MCF-7 cells were seeded into 6-well plates at a density of 1 × 10⁵ cells per well and cultured overnight to allow attachment. The following day, the cells were treated with various concentrations of 3ai or with 0.1% DMSO (vehicle control). The cells were then treated with the drug concentrations for 24 h, after which the culture medium was removed and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 3% BSA in PBS for 30 min at room temperature to prevent non-specific binding. Subsequently, the cells were incubated overnight at 4°C in the dark with the corresponding primary antibody. After washing, the cells were incubated with the fluorescent secondary antibody, diluted in 1% BSA, at room temperature in the dark. Nuclei were counterstained with DAPI for 15 min at room temperature in the dark. Finally, the samples were mounted with an anti-fade mounting medium, and images were acquired using a fluorescence microscope.

Flow cytometry analysis of cell cycle distribution

MCF-7 cells were treated with 3ai or vehicle control, then fixed overnight at -20°C in ice-cold 70% ethanol. The cells were then washed twice with PBS and incubated with 50 μg/mL propidium iodide for 30 min. Flow cytometric analysis was performed using a NovoCyte 2060R (Agilent Technologies, Santa Clara, CA, USA), with a minimum acquisition threshold of 20,000 events per sample. The distribution of cells across the G0/G1, S, and G2/M phases was analyzed.

Senescence-associated β-galactosidase staining

The cells were seeded into 6-well plates at a density of 1 × 10⁵ cells per well and cultured overnight. The cells were then treated with different concentrations of 3ai for 36 h. Senescence-associated β-galactosidase (SA-β-gal) activity was assessed using a SA-β-gal staining kit (Beyotime, C0602) according to the manufacturer’s instructions. SA-β-gal-positive staining was then visualized using light microscopy and quantified using ImageJ software.

Establishment of MCF-7 cells stably expressing siTRBP

293T cells were seeded at 5 × 10⁵ cells per well in 6-well plates and cultured to 80-90% confluence. For transfection, 3 μg pLKO.1-shTRBP, 0.75 μg pMG2D, and 2.25 μg psPAX were diluted in 250 μL Opti-MEM, mixed gently, and incubated at room temperature for 15-20 min. The mixture was added dropwise to each well and evenly distributed by gentle swirling. After 6-8 h, the medium was replaced with 2 mL serum-free medium. Viral supernatants were collected 48 h post-transfection, centrifuged at 1000 rpm for 3 min, filtered through a 0.45 μm membrane, and stored at -80°C. A second harvest was collected 24 h later. For infection, MCF-7 cells were seeded at 1 × 10⁵ cells per well 24 h before infection. On the day of infection, 1 mL viral supernatant was mixed with 1 mL serum-free medium containing Polybrene (8 μg/mL). The culture medium was removed and replaced with 1 mL of the mixture. After 24 h, the infection medium was replaced with 2 mL complete medium. Puromycin was added at a predetermined concentration 24 h post-infection for stable selection, with the medium refreshed every 2 d for 5-7 d until all negative control cells had died. A polyclonal pool of stable knockdown cells was thus established.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9 software. All experimental data are presented as mean ± standard deviation (SD), based on at least three independent biological replicates. Intergroup comparisons were conducted using one-way analysis of variance (ANOVA) for multiple group comparisons. *P < 0.05, **P < 0.01, ***P < 0.001 denote levels of statistical significance.

Results

3ai downregulates miR-21 by inhibiting its biogenesis

We used a previously established HeLa-luciferase-miR-21 reporter cell (Figure 1A) to screen a library of 202 newly synthesized small molecules. Using the previously reported miRNA-biogenesis modulator CIB-3b as a control, we screened dihydrocoumarin derivatives and identified a 3,4-diamino-substituted dihydrocoumarin, designated as 3ai (Figure 1B). Its chemical structure is shown in Figures 1C and S1. Compound 3ai increased luciferase activity in HeLa-luciferase-miR-21 cells-indicative of inhibition of endogenous miR-21 expression-with an EC₅₀ of 9.309 μM (Figure 1D). Accordingly, we selected 3ai for further study.

Figure 1.

3ai downregulates miR-21 by inhibiting its biogenesis. A. HeLa-luciferase-miR-21 reporter gene assay model for screening miR-21 small molecule inhibitors. B. Screening results for the inhibitory activity of 202 novel chemical compounds (10 μM) against miR-21, with red dots representing CIB-3b as the control and blue dots representing the dihydrocoumarin compound 3ai. C. Chemical structure of compound 3ai. D. HeLa-luciferase-miR-21 cells were exposed to increasing concentrations of 3ai for 24 h, after which luciferase reporter gene activity was measured. The EC50 value was calculated using GraphPad Prism. E. MCF-7 cells were treated with 3ai (10, 25 μM) for 24 h, and the expression levels of pre-miR-21 and mature miR-21 were measured by qRT-PCR. F, G. Western blot analysis was performed to evaluate the protein expression levels of PTEN and PDCD4. Data are presented as mean ± SD from triplicate experiments. Statistical significance was determined by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO control).

To validate the functional consequences of miR-21 inhibition, MCF-7 breast cancer cells were treated with graded concentrations of 3ai for 24 h, and endogenous miR-21 was quantified by qRT-PCR. 3ai significantly reduced miR-21 levels in MCF-7 cells, consistent with the luciferase reporter results. To further investigate the effect of 3ai on the miR-21 biogenesis, we measured the expression of the miR-21 precursor (pre-miR-21), and found that it was significantly increased. These data indicate that 3ai inhibits the processing of pre-miR-21 to mature miR-21 without affecting upstream steps, resulting in decreased mature miR-21 and accumulation of pre-miR-21 (Figure 1E). We next examined canonical miR-21 target genes. As shown in Figure 1F, 1G, the inhibition of miR-21 by 3ai resulted in marked upregulation of the validated targets PTEN and PDCD4.

3ai inhibits miRNA maturation by targeting TRBP

Our prior results showed that 3ai inhibits miR-21 maturation. MiRNA maturation involves the coordinated action of TRBP, Dicer, and Ago2, which together constitute the RISC-loading complex. This complex mediates the stepwise processing of precursor miRNAs to generate mature miRNAs. We therefore assessed the effect of 3ai on the expression of RISC-loading components by Western blotting. 3ai markedly reduced TRBP without significantly affecting Dicer or AGO2 (Figure 2A). These findings suggest that 3ai modulates miRNA biogenesis by selectively targeting TRBP. We next used the cellular thermal shift assay (CETSA) to test whether 3ai directly engages TRBP. Relative to the DMSO control, 3ai decreased TRBP thermal stability at 45°C (Figure 2B, 2C). In MCF-7 cell lysates, TRBP band intensity decreased in a dose-dependent manner with 3ai treatment (Figure 2D). We performed qRT-PCR analysis. The results demonstrated that 3ai treatment significantly decreased TRBP mRNA levels in MCF-7 cells, indicating that 3ai suppresses TRBP expression primarily at the transcriptional level (Figure 2E). Additionally, SPR analysis on a BIAcore CM5 chip was used to evaluate the interaction between 3ai and TRBP. Sensorgrams showed concentration-dependent responses upon compound titration, and kinetic analysis yielded a dissociation constant (KD) of 7.498 μM (Figure 2F). These results support a direct interaction between 3ai and TRBP.

Figure 2.

Analysis of the binding between 3ai and TRBP. (A) Western blot analysis of the expression levels of Dicer, Ago2, and TRBP in MCF-7 cells treated with 3ai (10 μM) for 24 h. (B) CETSA assay evaluating the effect of 3ai (10 μM) on the stability of Dicer and Ago2 in MCF-7 cell lysates at different temperatures. The stabilizing effect of compound 3ai on TRBP protein was assessed using cellular thermal shift assays (CETSA) in MCF-7 lysates under varying temperatures (C) and concentrations (D). GAPDH served as the loading control for normalization, with relative protein levels quantified through grayscale analysis (expressed as percentage values). All experiments were performed in triplicate with data presented as mean ± SD. Complementary validation through SDS-PAGE analysis with Coomassie staining confirmed the compound’s dose-dependent stabilization of TRBP in the lysate system. (E) MCF-7 cells were treated with 3ai (1, 10, 25 μM) for 24 h, and the mRNA expression levels of TRBP were measured by qRT-PCR. (F) SPR analysis using Biacore CM5 chip with recombinant TRBP protein immobilized to detect the binding of 3ai to TRBP protein. The KD was obtained from the affinity fitting curve.

3ai modulates the biogenesis of additional miRNAs

Western blotting and CETSA confirmed that 3ai selectively targets TRBP. Building on these findings, we profiled endogenous miRNA expression and found that 3ai reduced the levels of several mature miRNAs, consistent with impaired biogenesis (Figure 3A-C). A heatmap shows the enriched signaling pathways associated with the selected miRNAs. Pathways related to tumor progression (e.g., TGF-β signaling, p53 signaling, cell cycle, NF-κB signaling, and mTOR signaling) are prominently represented. In addition, RNA metabolism-related pathways, including RNA degradation and mRNA surveillance, are enriched. Notably, cellular senescence is also highlighted, suggesting a close association between the identified miRNAs and processes involving tumor development, RNA stability, and senescence regulation. The color scale indicates the significance level (-log₁₀ P value) (Figure 3D). Furthermore, the modulation of key cell cycle regulators, including the activation of p53, supports the induction of cellular senescence, as p53 plays a pivotal role in enforcing cell cycle arrest and promoting senescence under stress conditions. Previous studies have shown that the upregulation of let-7d and miR-335 could inhibit cellular senescence; miR-21 may regulate the cell cycle proteins, such as p21, to participate in the process of astrocyte senescence.

Figure 3.

3ai regulated of the biosynthesis of other miRNAs. A-C. MCF-7 cells were treated with 3ai (10 μM) for 24 h, and the expression of pre-miRNAs and mature miRNAs was detected by qRT-PCR. D. Functional enrichment analysis of miRNAs was performed using the miRPath database. E. MCF-7 cells were treated with si-TRBP, and the expression of other miRNAs was detected by qRT-PCR. F, G. MCF-7 cells were transfected with si-TRBP, and cell senescence status was assessed 36 hours later through β-galactosidase staining. H. Western blot analysis of p21 and TRBP expression in MCF-7 cells transfected with siNC or siTRBP (#1 and #2). GAPDH was used as a loading control. All data are presented as mean ± SD of triplicate experiments (*P < 0.05, **P < 0.01 and ***P < 0.001 compared to the DMSO).

3ai significantly downregulated the expression of let-7d and miR-335, a change expected to promote cellular senescence. Together, these data suggest that 3ai exerts antitumor activity by targeting TRBP to modulate the biogenesis of senescence-associated miRNAs and the expression of senescence-related genes, thereby inducing senescence in breast cancer cells. Consistent with the 3ai treatment, compared with the control group, the relative expression levels of multiple miRNAs (such as miR-22, etc.) in the si-TRBP group exhibited significant changes (Figure 3E). Moreover, senescence staining results showed that MCF-7 cells treated with siTRBP displayed more obvious senescent characteristics compared with the DMSO group (Figure 3F, 3G). After siTRBP treatment, the TRBP protein was decreased, and the expression of senescence-associated protein p21 was significantly induced (Figure 3H). Collectively, these results indicated that the regulatory effect of siTRBP on miRNAs was similar to that of 3ai treatment. Meanwhile, detected by senescence staining technology, MCF-7 cells treated with siTRBP showed obvious senescent characteristics, with a significant enhancement in the degree of cellular senescence.

High TRBP expression is associated with breast cancer development and progression

TRBP overexpression has been reported to disrupt miRNA biogenesis, thereby promoting tumor-cell proliferation and metastasis. Analysis of TRBP expression across tumor cell lines in the Human Protein Atlas database indicated that expression was highest in MCF-7 breast cancer cells (Figure 4A). To further validate TRBP expression, we profiled hepatocellular carcinoma (HCC) cell lines-MHCC97L (low metastatic potential), SK-HEP-1 (high metastatic potential) and HCCLM3 (high metastatic potential), lung cancer cell lines A549 and bleomycin (BLM)-induced A549 (a classical phenotype of senescent cells) [27], and breast cancer cell lines MCF-7 and MDA-MB-231 (high metastatic potential). Western blotting showed that TRBP levels in MCF-7 and MDA-MB-231 cells were significantly higher than those in the other cancer cell lines (Figure 4B, 4C), consistent with the database analysis. In addition, TRBP expression was reduced in BLM-induced senescent A549 cells, suggesting an association between lower TRBP levels and cellular senescence. Additionally, we analyzed TCGA breast cancer datasets, which revealed that TRBP mRNA expression was significantly higher in breast cancer tissues than in normal breast tissues (Figure 4D). Furthermore, analysis using KMplot (http://kmplot.com/analysis/) revealed that higher TRBP expression in patients with breast cancer was associated with worse survival (Figure 4E) and correlated with pathological stage (P < 0.05). These findings support TRBP as a therapeutic target in breast cancer (Figure 4F).

Figure 4.

High expression of TRBP in breast cancer and its impact on the survival of breast cancer patients. A. Expression levels of TRBP in different tumor cells in the database. B, C. Western validation of TRBP expression in different tumor cells. (BLM, MCE). D. Expression of TRBP in breast cancer (BRCA) based on sample types from TCGA database. E. The relationship between TRBP expression and the prognosis of breast cancer patients. F. The relationship between TRBP expression and the pathological staging of breast cancer tissues.

3ai suppresses proliferation and metastasis in breast cancer cells

Our previous results indicated that 3ai inhibits miR-21 biogenesis by targeting TRBP. MiR-21 and TRBP function as an oncogenic miRNA and an oncogene, respectively, and their overexpression is strongly associated with breast cancer progression. We therefore evaluated the antitumor effects of 3ai in breast cancer. CCK-8 assays showed that 3ai was non-toxic to normal breast epithelial MCF-10A cells but exhibited strong antiproliferative effects against the breast cancer cell lines MDA-MB-231 and MCF-7 (Figure 5A, 5B). In addition, 3ai inhibited MCF-7 and MDA-MB-231 colony formation in a dose-dependent manner (Figure 5C, 5D). Furthermore, 3ai inhibited MCF-7 and MDA-MB-231 migration (Figure 6A, 6B) and suppressed the epithelial-mesenchymal transition (EMT) by increasing the expression of the migration-related key protein E-cadherin and reducing the expression of N-cadherin (Figure 6C-F). Cell cycle analysis further revealed that 3ai induced G2-phase arrest (Figure 6G).

Figure 5.

3ai inhibits the proliferation of breast cancer cells. A. MCF-10A cells were treated with varying concentrations of 3ai for 24 h. Cell viability was assessed using the cell counting kit-8 (CCK-8) assay. B. MCF-7 and MDA-MB-231 cells were treated with 3ai (1, 10, 25 μM) for 24 h. Cell viability was measured by CCK-8. C, D. MCF-7 and MDA-MB-231 cells were treated with various concentrations of 3ai for 7-14 days, and colony formation was visualized by crystal violet staining and quantified. Data are presented as mean ± SD from triplicate experiments. Statistical significance was determined by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO control).

Figure 6.

3ai inhibits the migration of breast cancer cells. A, B. MCF-7 and MDA-MB-231 cells were treated with 3ai (1, 10, 25 μM) for 24 h, and cell migration was assessed via transwell assay. 10× magnification. Scale bar: 100 μm, n = 3×; C, D. MCF-7 cells were treated with 3ai for 48 h, and the expression of EMT markers was evaluated by western blot analysis. E, F. MDA-MB-231 cells were treated with 3ai for 48 h, and the expression of EMT markers was evaluated by western blot analysis. GAPDH was used as a loading control. G. Flow cytometric analysis of cell cycle distribution in MCF-7 cells treated with 3ai. Representative histogram data and statistical results are shown. Data are presented as mean ± SD from triplicate experiments. Statistical significance was determined by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO control).

3ai suppresses breast cancer cell proliferation by inducing senescence

Cell cycle arrest is a hallmark of senescence, and given our previous finding that 3ai-regulated miRNAs influence this process, we hypothesized that 3ai exerts antitumor effects in breast cancer by inducing senescence. To further examine the effects of 3ai on the cell cycle and cellular senescence, we performed western blotting to assess senescence-associated proteins p16, p21 and p53. 3ai increased expression of p16, p21 and p53 (Figure 7A-D). Senescence induction was further evaluated by β-galactosidase staining. 3ai induced senescence in breast cancer cells in a dose-dependent manner (Figure 7E, 7F). To further confirm the induction of cellular senescence, we examined the expression of Ki67, a well-established marker of proliferation and senescence, using immunofluorescence analysis. The results showed that 3ai markedly reduced the expression of Ki67 (Figure 7G, 7H). Western blot analysis revealed that 3ai elevated the level of the DNA damage marker γ-H2A.X, suggesting persistent DNA damage, a hallmark feature of cellular senescence (Figure 8A, 8B). Furthermore, we investigated the effect of 3ai on MMP1, a SASP factor implicated in tumor invasion and metastasis. 3ai significantly suppressed MMP1 expression (Figure 8C, 8D). We performed Western blotting analysis to evaluate the energy metabolism marker AMP-activated protein kinase (AMPK) in senescent cells. The results demonstrated that treatment with 3ai markedly increased the phosphorylation level of AMPK. Moreover, 3ai inhibited the downstream mammalian target of rapamycin complex 1 (mTORC1) signaling pathway through AMPK activation (Figure 8E-H). These findings indicate that 3ai induces senescence in breast cancer cells by modulating energy metabolism through the AMPK-mTOR signaling pathway.

Figure 7.

3ai induces cellular senescence in breast cancer cells. A, B. Treat MCF-7 cells with different concentrations of 3ai for 48 h, and then measure the expression levels of senescence-associated proteins p16, p21, p53 by western blot. C, D. Treat MDA-MB-231 cells with different concentrations of 3ai for 48 h, and then measure the expression levels of senescence-associated proteins p16, p21, p53 by western blot. E, F. Treating MCF-7 and MDA-MB-231 cells with varying concentrations of 3ai, cell senescence status was assessed 36 h later through β-galactosidase staining. 20× magnification. Scale bar: 100 μm, n = 3×. G, H. Treat MCF-7 cells with 3ai (10 μM) for 48 h, and then detect the expression change of Ki67 by immunofluorescence staining. 20× magnification. Scale bar: 100 μm, n = 3×. Data are presented as mean ± SD from triplicate experiments. Statistical significance was determined by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO control).

Figure 8.

Effect of 3ai treatment on the expression of key proteins. A, B. Treat MCF-7 cells with different concentrations of 3ai for 48 h, and then measure the expression levels of γ-H2A.X by western blot. C, D. Treating MCF-7 cells with different concentrations of 3ai, changes in the expression levels of MMP1 by western blot 48 h later. E, F. Treating MCF-7 cells with different concentrations of 3ai, the expression levels of the AMPK/mTOR by western blot. G, H. Treating MDA-MB-231 cells with different concentrations of 3ai, the expression levels of the AMPK/mTOR by western blot. GAPDH was used as a loading control. Data are presented as mean ± SD from triplicate experiments. Statistical significance was determined by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO control).

Discussion

TRBP (trans-activation response RNA-binding protein 2, TARBP2) is a highly conserved double-stranded RNA-binding protein (dsRBP) that plays a key role in the RNA interference (RNAi) pathway [28]. As a core component of the RNA-induced silencing complex (RISC), TRBP cooperates with Dicer to cleave precursor microRNAs (pre-miRNAs) and promote the accurate loading of miRNA strands onto Argonaute 2 (AGO2), thereby facilitating miRNA biogenesis and target mRNA degradation, ultimately regulating gene expression [22]. In breast cancer, TRBP promotes the maturation of oncogenic miRNAs (such as miR-21 and miR-155) and represses tumor-suppressor genes (such as PTEN and PDCD4), thereby promoting the proliferation and metastasis of breast cancer cells [29].

A previous study has shown that the benzoxazole-based small molecule CIB-3b [25] allosterically modulates TRBP, thereby influencing miRNA expression and highlighting TRBP as a therapeutic target in oncology. In this study, we screened 202 structurally novel small-molecules using the same cellular model employed for CIB-3b. We identified a highly active 3,4-diamino-substituted dihydrocoumarin, designated 3ai, that targets and inhibits TRBP, thereby regulating the biosynthesis of multiple miRNAs, including miR-21. Unlike CIB-3b, which does not inhibit TRBP expression but interferes with the TRBP-Dicer interaction (thereby affecting precise cleavage and maturation of miRNAs), our results in Figure 2 demonstrate that 3ai not only suppresses TRBP expression but also physically binds to TRBP. Based on these findings, we hypothesize that 3ai may disrupt TRBP’s phosphorylation and ubiquitination modifications through direct binding, ultimately influencing its degradation. In future studies, we will investigate the binding sites of 3ai on TRBP and its effects on TRBP’s post-translational modifications.

Furthermore, we confirmed TRBP overexpression in breast cancer tissues and demonstrated a significant association with poor patient prognosis. This evidence establish TRBP as a potential novel therapeutic target in breast cancer. Our subsequent studies demonstrate that 3ai effectively suppresses breast cancer cell proliferation and metastasis (Figures 5, 6) by specifically inhibiting TRBP-mediated miRNA biogenesis (Figure 3). Functional enrichment analysis of 3ai-regulated miRNAs targeting TRBP revealed their significant involvement in cellular senescence pathways. Consequently, we systematically evaluated the effects of 3ai on breast cancer cells. As shown in Figure 7, 3ai treatment markedly enhanced β-galactosidase staining in breast cancer cells and upregulated classical senescence markers (p16, p21, and p53) [30,31].

Cellular senescence serves as a critical tumor-suppressive mechanism, primarily mediated through permanent cell cycle arrest [32]. Senescent cells produce a senescence-associated secretory phenotype (SASP), which exerts paracrine effects that induce senescence in neighboring cells, thereby establishing a tumor-inhibitory barrier that suppresses cancer cell proliferation and metastasis [33]. Dysregulation of post-transcriptional control contributes to numerous complex diseases, with aberrant regulation of mRNA stability playing a significant role in both cellular senescence and tumorigenesis [34]. Notably, studies have demonstrated that TRBP can bind to the overexpressed structural RNA stability element (sRSE) in highly migratory cells, such as breast cancer, where it destabilizes metastasis-suppressor mRNAs and consequently promotes cancer cell invasion and metastasis [35,36]. Based on these findings, we propose that TRBP functions as a core regulatory RNA-binding protein (RBP) that controls both miRNA maturation and mRNA stability through RBP-RNA interactions. This complex regulatory network profoundly influences cell growth and proliferation and plays a pivotal role in the post-transcriptional regulation of senescence-related genes. AMP-activated protein kinase (AMPK) is a central regulator of energy metabolism and proteostasis that also influences senescence [37,38]. Western blotting revealed a marked increase in AMPK phosphorylation following 3ai treatment. AMPK activation is known to inhibit mammalian target of rapamycin complex 1 (mTORC1) signaling, thereby promoting senescence [39,40].

In addition, dihydrocoumarin has been reported to act as a sirtuin (SIRT) inhibitor, promoting cellular senescence and apoptosis by inhibiting SIRT1 [41]. Although some studies have reported inhibitory effect of dihydrocoumarin derivatives on breast cancer cells activity, the underlying mechanisms remain poorly understood [42]. As a novel dihydrocoumarin derivative, 3ai targets TRBP to regulate miRNAs biogenesis, thereby inducing cellular senescence through multiple pathways-including cell cycle checkpoint activation and DNA damage induction-and ultimately suppressing the proliferation and metastasis of breast cancer cells (Figure 9).

Figure 9.

Schematic diagram illustrating the mechanism by which 3ai targets and inhibits TRBP, induces senescence, and suppresses breast cancer progression.

Conclusion

In summary, we identify 3ai as a novel dihydrocoumarin derivative that selectively targets TRBP, thereby inhibiting miRNA biogenesis and blocking the maturation of oncogenic miR-21. By inducing cellular senescence-mediated by cell cycle arrest, DNA damage, and AMPK/mTORC1 pathway regulation, 3ai effectively inhibits the proliferation and metastasis of breast cancer cells. These findings highlight TRBP as a critical regulator of tumor progression and establish 3ai as a promising lead compound for developing TRBP-targeted cancer therapeutics. While our findings establish 3ai as a TRBP-targeted lead, rigorous in vivo validation-including pharmacokinetics, exposure - efficacy relationships, and safety margins-remains a critical next step to substantiate translational potential.

Disclosure of conflict of interest

None.