8-Chloro-adenosine inhibits breast cancer progression by inducing ferroptosis via the ADAR1/miR-101-3p/SLC7A11 axis.

Introduction
Breast cancer is the most frequently diagnosed cancer and a leading cause of cancer-related mortality among women worldwide. According to the International Agency for Research on Cancer (IARC), approximately 2.3 million new breast cancer cases were diagnosed in 2022, making it the second most common cancer globally [1]. Despite advances in screening, diagnosis, and treatment, the incidence of breast cancer continues to rise, particularly in high-income countries [2]. Moreover, limited access to effective diagnostics and therapeutic resources in low- and middle-income regions exacerbates global health disparities, underscoring the urgent need for novel and more effective therapeutic strategies.
Ferroptosis, a novel form of regulated cell death characterized by iron-dependent lipid peroxidation [3–5], first proposed by Stockwell in 2012 [6]. Unlike apoptosis or necroptosis, ferroptosis involves the accumulation of reactive oxygen species (ROS) accumulation and lipid peroxidation-induced oxidative damage, and is tightly regulated by intracellular iron metabolism and antioxidant systems. Labile iron catalyzes the Fenton reactions with hydrogen peroxide (H₂O₂) to generate excessive ROS, ultimately triggering ferroptotic cell death via oxidative damage [7]. Solute carrier family 7 member 11 (SLC7A11/xCT), the cystine/glutamate antiporter that imports cystine for glutathione (GSH) synthesis, is a key negative regulator of ferroptosis [8]. High SLC7A11 expression occurs across multiple tumor types [9], and its inhibition disrupts GSH biosynthesis, inactivates glutathione peroxidase 4 (GPX4), and triggers lipid peroxidation-driven ferroptotic death. Targeting SLC7A11 thus represents a promising anticancer strategy. Although ferroptosis contributes to tumor suppression and therapy response in breast cancer [10, 11], its precise regulatory mechanisms remain elusive and warrant further study.
8-Chloro-adenosine (8-Cl-Ado), a synthetic adenosine analogue, exerts potent anti-tumor effects in hematological and solid malignancies [12–14] and is currently under Phase I evaluation for acute myeloid leukemia (AML) [12, 15, 16]. Intracellularly, 8-Cl-Ado can be metabolized to 8-chloro-ATP (8-Cl-ATP), which functions as a chain terminator in RNA synthesis, ultimately inducing energy depletion and cell death [13, 15, 17]. We previously demonstrated that 8-Cl-Ado effectively suppresses breast cancer cell proliferation, migration, and invasion by targeting the RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) [18–20]. ADAR1 catalyzes the conversion of adenosine to inosine within double-stranded RNA and plays important roles in RNA metabolism, immune response, and cancer progression [21–24]. We further reported that 8-Cl-Ado downregulates ADAR1 expression in breast cancer cells by suppressing the transcription factor PU.1, which binds to the promoter region of the ADAR1 gene [25]. Whether 8-Cl-Ado triggers ferroptosis and the role of ADAR1 in this context remain to be elucidated.
In this study, we investigated the role of 8-Cl-Ado in regulating ferroptosis in breast cancer both in vitro and in vivo. We identified a novel pathway by which 8-Cl-Ado induces ferroptosis to inhibit breast cancer progression via ADAR1/miR-101-3p/SLC7A11 signaling axis. Furthermore, we demonstrated that ADAR1 impairs the maturation of miR-101-3p by binding to its precursor RNA (pre-miR-101-2) in an RNA-editing-independent manner. These findings provide novel mechanistic insights into the anti-tumor effects of 8-Cl-Ado and offer a potential therapeutic strategy for enhancing ferroptosis by targeting ADAR1 in breast cancer therapy.

Materials and methods

Cell lines, cell culture and drug treatment
Human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; VivaCell, Shanghai, China) and 1% penicillin-streptomycin (Beyotime, Shanghai, China), and maintained at 37 °C in a humidified incubator with 5% CO2. 8-Chloro-adenosine (8-Cl-Ado) was purchased from BIOLOG life Science Institute (Bremen, Germany) and dissolved in sterile phosphate-buffered saline (PBS) to prepare a stock solution at an appropriate concentration.

Plasmids, MiRNA mimics, anti-sense oligonucleotide (ASO) and transfection
Wild-type ADAR1 (WT) overexpression plasmids, including ADAR1-p150 and ADAR1-p110 isoforms, the editing-deficient mutant overexpression plasmid (Mut, ADAR1-p150 ΔE/A; ΔE/A denotes editing-deficient mutant), and CRISPR/Cas9 plasmids (Ctrl sgRNA, which does not target any sequence, and ADAR1 sgRNA, which specifically targets ADAR1) were previously described in our studies [26–28]. MiR-101-3p mimics, corresponding negative control mimics (NC) and miR-101-3p anti-sense oligonucleotide (ASO) were purchased from Shanghai GeneBio Co.,Ltd (Shanghai, China). Plasmids, miRNA mimics and miRNA ASO were transfected using Lipofectamine 2000 (Lipo2000; CYTOCH, Shanghai, China) according to the manufacturer’s instructions, when cell confluence reached approximately 70–80% in 6-cm dishes or 6-well plates. The miR-101-3p mimics and ASO sequences were listed in the Table S1.

Cell viability assay
Cell viability was determined using the Cell Counting Kit-8 (CCK-8; Beyotime, Shanghai, China). MCF-7 and MDA-MB-231 cells were seeded into 96-well plates at a density of 2 × 10³ cells per well in 100 µL of complete medium. After treatment, 10 µL of CCK-8 reagent was added to each well, followed by incubation at 37 °C for 1 h. The absorbance at 450 nm was measured using a microplate reader (Bio-Tek, Rockville, MA, USA).

5-Ethynyl-2'-Deoxyuridine (EdU) assay
Cell proliferation was assessed using the EdU cell proliferation assay kit (C0071S, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Following transfection or 8-Cl-Ado treatment, MCF-7 and MDA-MB-231 cells were seeded into 6-well plates. A 10 µM EdU working solution was prepared and added to the wells, and the cells were incubated at 37 °C for 2 h. The percentage of EdU-positive cells was determined using a fluorescence microscope (Leica, Wetzlar, Germany).

Dual-luciferase reporter assay
Wild-type (WT) and mutant (Mut) reporter constructs, containing the SLC7A11 mRNA 3′UTR sequence predicted to be targeted by miR-101-3p, were generated using the pmirGLO dual-luciferase reporter vector (GENE CREATE, Wuhan, China) and verified by sequencing (Fig. S1). The sequences for SLC7A11-3′UTR-WT and SLC7A11-3′UTR-Mut are shown in Fig. S1. The SLC7A11 3′UTR sequence was cloned into the NheⅠ and XhoⅠ restriction sites within the multiple cloning site (MCS) of the pmirGLO dual-luciferase reporter vector (Fig. S1). Both the WT and Mut SLC7A11 3′UTR DNA fragments were synthesized by GENE CREATE (Wuhan, China). MCF-7 and MDA-MB-231 cells were co-transfected with either WT or Mut reporter plasmids, together miR-101-3p mimics or negative control (NC) mimics. After 48 h, cells were collected, and luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Beyotime, Shanghai, China). Firefly luciferase activity was normalized to Renilla luciferase activity as an internal control.

Migration and invasion assay
Cell migration and invasion were evaluated using Transwell chambers (8.0 μm pore size; Beyotime, Shanghai, China). For the invasion assay, a Matrigel mixture (ABW, Shanghai, China) was prepared by diluting Matrigel with DMEM at a 1:8 (v/v) ratio. A total of 60 µL of this mixture was added to the upper chamber and incubated at 37 °C for 2 h to allow gel solidification. For the migration assay, no Matrigel was added to the upper chamber. Breast cancer cells (5 × 10⁴ cells per chamber) were suspended in serum-free medium and seeded into the upper chamber, while the lower chamber was filled with 600 µL of medium containing 20% FBS (VivaCell, Shanghai, China). After 48 h of incubation, non-migratory/non-invasive cells in the upper chamber were gently removed, and the remaining cells on the lower surface of the membrane were fixed with immunostaining fixative (Beyotime, Shanghai, China) for 30 min and stained with crystal violet (Beyotime, Shanghai, China) for 5 min. Three random fields per well were photographed and quantified using a light microscope (Leica, Wetzlar, Germany).

RNA extraction and qRT-PCR
Total RNA was extracted from designated cells using Trizol reagent (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. mRNA reverse transcription was performed using the ABScript III RT kit (ABclonal, Wuhan, China), while miRNA reverse transcription was carried out using the PrimeScript™ RT kit (Takara Bio, Japan). Quantitative real-time PCR (qRT-PCR) was conducted using a 2× Universal SYBR rapid qPCR mixture (Sangon Biotech, Shanghai, China) on the Bio-Rad CFX system. The qRT-PCR procedure was as described previously [27, 28]. GAPDH was used as an internal control for mRNA, and U6 was used as an internal control for miRNA normalization. Relative expression levels were calculated using the 2−ΔΔCt rule. All primer sequences used were listed in the Table S1.

Western blot analysis
Cells were lysed using RIPA lysates (Proteintech, Wuhan, China) and protein concentrations were determined using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific, Hercules, CA, USA) [26, 27]. Band intensities were quantified using ImageJ software (National Institutes of Health, NIH, USA). The antibodies used were as follows: mouse anti-ADAR1 (sc-73408, Santa Cruz Biotechnology, CA, USA), mouse anti-GAPDH (M20006, Abmart, Shanghai, China;), rabbit anti-GPX4 (T56959, Abmart, Shanghai, China), rabbit anti-SLC7A11 (T57046, Abmart, Shanghai, China).

Reactive oxygen species assay (ROS) assay
Intracellular reactive oxygen species (ROS) levels were assessed using the DCFH-DA fluorescent probe (S0033S, Beyotime, Shanghai, China). Following transfection or drug treatment, cells were incubated with DCFH-DA at 37 °C for 50 min in the dark, then stained with DAPI (C1002, Beyotime, Shanghai, China) at room temperature for 10–15 min to visualize nuclei. Stained cells were observed under a fluorescence microscope (Leica, Wetzlar, Germany). Nuclei exhibited blue fluorescence (DAPI), while ROS-positive cells emitted green fluorescence (DCFH-DA), with fluorescence intensity reflecting intracellular ROS accumulation.

Determination of intracellular reduced glutathione (GSH), iron (Fe2+) and malondialdehyde (MDA)
Cells were collected, resuspended in PBS (for Fe²⁺ and GSH detection) or RIPA buffer (for MDA detection), and lysed by ultrasonication. Intracellular GSH levels were quantified using a GSH assay kit (A006-2-1, Nanjing Jiancheng, Nanjing, China) according to the manufacturer’s instructions. Intracellular ferrous iron (Fe²⁺) concentration was measured with a ferrous ion detection kit (JL-T1255, Jianglaibio, Shanghai, China). Malondialdehyde (MDA) content, a marker of lipid peroxidation, was determined using an MDA assay kit (S0131S, Beyotime, Shanghai, China) following the manufacturer’s instructions.

Immunohistochemistry (IHC)
As described previously [28]. Briefly, the breast cancer tissues from the animal model were fixed with 4% paraformaldehyde for 48 h, dehydrated, and embedded in paraffin. Paraffin-embedded tissue sections were processed using the PV-9000 IHC kit (ZSGB-BIO, Beijing, China) according to the manufacturer’s instructions. Sections were incubated overnight at 4 °C with primary antibodies against ADAR1 (1:100, sc-73408, Santa Cruz, USA), SLC7A11 (1:100, T57046, Abmart, Shanghai, China), and GPX4 (1:100, T56959, Abmart, Shanghai, China). After washing with PBS, sections were incubated with HRP-conjugated secondary antibodies (anti-mouse or anti-rabbit) for 1 h at room temperature. Signals were developed using 3,3′-diaminobenzidine (DAB) substrate, followed by hematoxylin counterstaining. Finally, sections were mounted with neutral balsam and coverslips, and examined under a light microscope (Leica, Wetzlar, Germany).

Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization (FISH) assay was performed to detect the subcellular localization of pre-miR-101-2 and miR-101-3p. Cy3-labeled pre-miR-101-2 probes and FAM-labeled miR-101-3p probes were synthesized by Gene Pharma (Gene Pharma, Shanghai, China). MCF-7 and MDA-MB-231 cells were transfected with the ADAR1 overexpression plasmid, and 48 h later, cells were processed for FISH assay according to the manufacturer’s instructions. The probe working solution was prepared by mixing 2 µL of 1 µM biotin-labeled probe with 1 µL of 1 µM streptavidin-Cy3; the mixture was denatured and applied to the cells after equilibration. Hybridization was performed overnight at 37 °C. After washing, nuclei were counterstained with DAPI (Beyotime, Shanghai, China) at room temperature for 15 min. Fluorescence signals were visualized using a ZOE™ fluorescence microscope (BIO-RAD, USA). Probe sequences were listed in the Table S2.

RNA Immunoprecipitation (RIP) assay
MCF-7 and MDA-MB-231 cells were transfected with ADAR1 overexpression plasmids. After 48 h, cells were collected, washed with PBS, and lysed. Cell lysates were incubated overnight at 4 °C with 2 µg of anti-ADAR1 antibody (sc-73408, Santa Cruz Biotechnology, USA) or 2 µg of normal mouse IgG (A7028, Beyotime Biotechnology, Shanghai, China) as a negative control. Subsequently, 25 µL of pre-washed Protein A/G magnetic beads (P2108, Beyotime Biotechnology) were added to capture antibody/ADAR1/miRNA complexes. Co-precipitated RNAs were extracted using Trizol reagent and analyzed by qRT-PCR with specific primers. Primers were synthesized by BioTNT (Shanghai, China), and their sequences were listed in the Table S1.

In vivo model
SPF female nude mice aged 4–6 weeks were purchased from Chongqing ENSIWEIER Biotechnology (Chongqing, China) and housed in barrier facility cages. Breast cancer cells were subcutaneously injected into the flank. When the maximum tumor diameter reached approximately 3 mm, mice were administrated with 8-Cl-Ado (100 mg/kg, dissolved in PBS) or PBS via intraperitoneal injection every 3 days for 3 weeks [14]. Tumor size (diameter and volume), number, and distribution were monitored every 3 days. Mice were sacrificed within 1–3 days after the final injection, and the tumors were excised for further analysis. Tumor volume was calculated based on measured dimensions. All animal studies were approved by the Life Science Ethics Review Committee of Chongqing Medical University (Ethics Number: IACUC-CQMU-2024–0793).

Application of bioinformatics tools
The targeting correlation between miR-101-3p and SLC7A11 was analyzed using the ENCORI database (https://rnasysu.com/encori/). The schematic diagram was created with Figdraw (https://www.figdraw.com/static/index.html).

Statistical analysis
All experiments were repeated at least three times independently. Data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA). Comparisons between two groups were made using two-tailed Student’s t-tests, while multiple group comparisons were analyzed by one-way ANOVA. A p value < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Results

8-Cl-Ado induces ferroptosis in breast cancer cells
Accumulating evidence has demonstrated that 8-Cl-Ado exerts broad anti-tumor effects and can induce apoptosis in various types of cancers, including lung and breast cancer cells [18, 19, 29]. To further evaluate its role in breast cancer, MCF-7 and MDA-MB-231 breast cancer cell lines were exposed to 10 µM 8-Cl-Ado, and cell viability was assessed using the CCK-8 assay. As shown in Fig. 1A, 8-Cl-Ado significantly inhibited the proliferation of breast cancer cells in a time-dependent manner compared with the control group. EdU incorporation assays further confirmed that 8-Cl-Ado markedly reduced cell proliferation (Fig. 1B), while Transwell assays revealed significant inhibition of both migration and invasion (Fig. 1C).
To investigate whether the anti-tumor effects of 8-Cl-Ado are associated with ferroptosis, we examined key ferroptosis regulators and biochemical markers. Western blot analysis revealed a notable decrease in the expression of SLC7A11 and GPX4 in 8-Cl-Ado-exposured cells (Fig. 1D). Furthermore, 8-Cl-Ado significantly increased intracellular levels of Fe²+, malondialdehyde (MDA), and reactive oxygen species (ROS), while reducing glutathione (GSH) levels (Fig. 1E-H), compared with the respective control groups. Collectively, these data suggest that 8-Cl-Ado induces ferroptosis in breast cancer cells.

8-Cl-Ado inhibits breast cancer growth and downregulates SLC7A11 and GPX4 expression in vivo
To evaluate the in vivo efficacy of 8-Cl-Ado in breast cancer, we established MCF-7 xenograft models in nude mice as described in materials and methods section. Mice were administered 8-Cl-Ado intraperitoneally at a dose of 100 mg/kg every three days for three weeks (Fig. 2A). Tumor growth was significantly inhibited in the 8-Cl-Ado treatment group compared to controls, with notable differences observed from day 9 onward (Fig. 2B-D). qRT-PCR and Western blot analyses of tumor tissues revealed reduced mRNA and protein levels of SLC7A11 and GPX4 following 8-Cl-Ado treatment (Fig. 2E, F). Immunohistochemistry further confirmed the downregulation of SLC7A11 and GPX4 in tumor sections (Fig. 2G, Fig. S2), suggesting that 8-Cl-Ado could induce ferroptosis in vivo.

8-Cl-Ado induces ferroptosis by upregulating miR-101-3p to suppress SLC7A11 expression
To elucidate the mechanism by which 8-Cl-Ado induces ferroptosis, we focused on SLC7A11, a key regulator of ferroptosis [8, 9]. As previously shown (Figs. 1D and 2E–G), 8-Cl-Ado treatment markedly reduced SLC7A11 protein levels. To determine whether this reduction of SLC7A11 occurs at the transcriptional level, we performed qRT-PCR and observed a significant downregulation of SLC7A11 mRNA in 8-Cl-Ado-treated MCF-7 and MDA-MB-231 breast cancer cells (Fig. 3A). Given that 8-Cl-Ado is known to modulate microRNAs (miRNAs) in breast cancer [18], we hypothesized that its effect on SLC7A11 might be mediated by miRNAs. To identify potential miRNAs targeting SLC7A11 mRNA, we integrated miRNA array data from 8-Cl-Ado-treated MDA-MB-231 cells (fold change > 2) with predictions from the ENCORI and miRWalk databases. This analysis yielded two candidate miRNAs: miR-101-3p and miR-339-5p (Fig. 3B). Subsequent qRT-PCR validation revealed that miR-101-3p expression was significantly upregulated by 8-Cl-Ado treatment, whereas miR-339-5p showed only a modest change (Fig. 3C, D). Moreover, correlation analysis using the ENCORI database demonstrated a negative correlation between miR-101-3p and SLC7A11 expression (Fig. 3E). Based on these findings, we selected miR-101-3p for further investigation.
To functionally validate the role of miR-101-3p, we transfected MCF-7 and MDA-MB-231 cells with miR-101-3p mimics and assessed SLC7A11 expression. Both mRNA and protein levels of SLC7A11 were significantly reduced following miR-101-3p mimics transfection (Fig. 3F, G). We then predicted potential miR-101-3p binding sites within the 3′UTR of SLC7A11 mRNA using the ENCORI database (Fig. 3H). A dual-luciferase reporter assay was performed using wild-type (WT) and mutant (Mut) reporter constructs containing SLC7A11 mRNA 3′UTR sequence to confirm direct interaction between miR-101-3p and SLC7A11 mRNA. Co-transfection of miR-101-3p mimics with the WT construct significantly decreased luciferase activity, whereas the Mut construct showed no change (Fig. 3I). To confirm the role of miR-101-3p in mediating 8-Cl-Ado’s effect, we performed rescue experiments by transfecting cells with a miR-101-3p antisense oligonucleotide (ASO). The result showed that inhibition of miR-101-3p restored SLC7A11 protein expression in treated MCF-7 and MDA-MB-231 cells compared to their respective controls (Fig. 3J). These results confirm that miR-101-3p directly targets the 3′UTR of SLC7A11 mRNA, leading to its downregulation and possible subsequent induction of ferroptosis.

miR-101-3p induces ferroptosis in breast cancer cells
To investigate whether miR-101-3p alone could induce ferroptosis. We transfected miR-101-3p mimics into MCF-7 and MDA-MB-231 cells, followed by CCK-8 and EdU incorporation assays to detect cell proliferation. As shown in Fig. 4A and B, miR-101-3p mimics effectively inhibited the proliferation of breast cancer cells, compared to NC mimics control group. Subsequently, Transwell assays were conducted to detect cell migration and invasion. As shown in Fig. 4C, both migration and invasion were markedly reduced after transfection of miR-101-3p mimics into MCF-7 and MDA-MB-231 cells, compared to NC mimics control groups. Thus, miR-101-3p inhibited the proliferation, migration and invasion of breast cancer cells. Finally, we detected the levels of Fe2+, GSH, MDA and ROS after transfection of miR-101-3p mimics into MCF-7 and MDA-MB-231 cells. The results showed that the levels of Fe2+, MDA and ROS were increased, while the level of GSH was decreased after transfection of miR-101-3p-mimics, compared to NC mimics control groups (Fig. 4D-G), indicating that miR-101-3p induces ferroptosis in breast cancer cells.

8-Cl-Ado regulates miR-101-3p via ADAR1
To further elucidate how 8-Cl-Ado regulates miR-101-3p to induce ferroptosis, we explored the potential role of ADAR1. Our previous study demonstrated that 8-Cl-Ado inhibits ADAR1 transcription by modulating the transcription factor PU.1 [25]. Given that ADAR1 is an RNA-editing enzyme known to regulate gene expression post-transcriptionally by influencing the biogenesis of microRNAs [30, 31], we hypothesized that 8-Cl-Ado may modulate miR-101-3p expression via ADAR1.
To test this, we first examined the expression of ADAR1 following 8-Cl-Ado treatment. qRT-PCR and Western blot analyses revealed that treatment with 10 µM 8-Cl-Ado for 48 h significantly reduced both mRNA and protein levels of ADAR1 in MCF-7 and MDA-MB-231 breast cancer cell lines (Fig. 5A, B). Next, we assessed whether ADAR1 regulates miR-101-3p expression. ADAR1 gene knockout via CRISPR/Cas9 system was confirmed by sequencing (Fig. S3). Knockdown of ADAR1 led to a marked upregulation of miR-101-3p, while ADAR1 overexpression suppressed miR-101-3p levels in both cell lines (Fig. 5C, D). Consistently, in vivo analysis showed that 8-Cl-Ado treatment reduced ADAR1 expression and concomitantly increased miR-101-3p levels (Fig. 5E, F) in mouse tumor tissues described in Fig. 2.
MiR-101-3p is primarily derived from its precursor, pre-miR-101-2, which undergoes nuclear export and cytoplasmic processing by Exportin-5 and Dicer, respectively [32]. We speculated that ADAR1 may inhibit miR-101-3p maturation by interacting with pre-miR-101-2. To investigate this, we first assessed the cellular localization of pre-miR-101-2 and mature miR-101-3p following ADAR1 overexpression by using fluorescence in situ hybridization (FISH). The results revealed an increased accumulation of pre-miR-101-2 in ADAR1-overexpressing cells (Fig. 6A, B), and efficient ADAR1 overexpression in both MCF-7 and MDA-MB-231 cells was confirmed by western blotting (Fig. 6C). We next performed RNA immunoprecipitation (RIP) assays with an ADAR1 antibody to determine its binding to pre-miR-101-2 and mature miR-101-3p. The results demonstrated a significant enrichment of pre-miR-101-2, but not miR-101-3p, in the ADAR1 immunoprecipitate (Fig. 6D-G). Notably, this interaction with pre-miR-101-2 was observed for both wild-type ADAR1 (ADAR1-p150 and ADAR1-p110 isoforms) and an RNA-editing-deficient mutant (ADAR1-EA), indicating that the RNA-editing activity of ADAR1 is dispensable for this regulatory interaction. These results suggest that ADAR1 interacts with pre-miR-101-2, thereby impairing its maturation into the miR-101-3p.

8-Cl-Ado regulates ferroptosis via the ADAR1/miR-101-3p/SLC7A11 axis
Based on our preliminary findings, we asked whether 8-Cl-Ado induces ferroptosis via the ADAR1/miR-101-3p/SLC7A11 axis and whether ADAR1 functions in this process independently of its RNA-editing activity. To this end, we manipulated ADAR1 expression in breast cancer cells with or without 8-Cl-Ado treatment, and assessed ferroptotic phenotypes, key markers, and the expression levels of the axis components. MCF-7 and MDA-MB-231 cells were transfected with either an ADAR1-sgRNA plasmid or an ADAR1-overexpression plasmid (p150, p110, or editing-inactive mutant ADAR1-ΔE/A), followed by treatment with 10 µM 8-Cl-Ado for 48 h. Cell viability and proliferation were assessed using CCK-8 assays (Fig. S4A, B) and EdU incorporation assays (Fig. S4C, D), while cell migration and invasion were detected using Transwell assays (Fig. S4E, F). As expected, 8-Cl-Ado significantly suppressed cell proliferation, migration and invasion. These inhibitory effects were further enhanced by ADAR1 knockdown and partially reversed by ADAR1 overexpression.
To evaluate the induction of ferroptosis, we measured intracellular levels of GSH, Fe2+, MDA, and ROS. The results showed that ADAR1 knockdown or 8-Cl-Ado treatment significantly increased Fe2+, MDA, and ROS levels, while decreasing GSH content (Figs. 7A-D and 8A-D), consistent with ferroptosis activation. These ferroptotic changes were enhanced by ADAR1 knockdown and attenuated by overexpression of ADAR1 isoforms, including the RNA-editing-deficient mutant ADAR1-ΔE/A, suggesting that ADAR1 inhibits ferroptosis in a manner independent of its editing activity.
Western blot analysis further confirmed that 8-Cl-Ado treatment reduced ADAR1, SLC7A11, and GPX4 protein levels, and this effect was augmented by ADAR1 knockdown but reversed by overexpression of ADAR1 isoforms (Figs. 7E and 8E). Notably, both wild-type (p150, p110) and editing-inactive ADAR1-ΔE/A isoforms restored SLC7A11 and GPX4 expression, supporting a regulatory mechanism independent of RNA editing activity.
Taken together, these results demonstrate that 8-Cl-Ado induces ferroptosis in breast cancer cells by downregulating ADAR1, which in turn relieves its suppression on miR-101-3p maturation. miR-101-3p subsequently targets and represses SLC7A11, thereby triggering ferroptosis. Importantly, this regulatory effect of ADAR1 appears to be independent of its RNA-editing activity.

Discussion
In this study, we demonstrate that 8-Cl-Ado exerts anti-tumor effects in breast cancer by inducing ferroptosis through the ADAR1/miR-101-3p/SLC7A11 signaling axis (Fig. 9). 8-Cl-Ado treatment led to elevated intracellular lipid ROS, Fe²+, and MDA levels, reduced GSH contents, and downregulation of SLC7A11, a key regulator of ferroptosis. Notably, ADAR1 binds with pre-miR-101-2 to hinder the formation of mature miR-101-3p, and RNA-editing activity of ADAR1 is not required for this process. Our findings uncover a novel anti-tumor mechanism of 8-Cl-Ado and provide important insights into ferroptosis-based therapeutic strategies for breast cancer.
Previous studies have demonstrated that 8-Cl-Ado, a C8-modified adenosine analog, exhibits cytotoxicity in solid tumor cells [33]. A recent study demonstrated that 8-Chloro-adenosine induces apoptotic cell death in cholangiocarcinoma cells [34]. Intracellularly, 8-Cl-Ado is phosphorylated by adenosine kinase to its active triphosphate form, which disrupts RNA metabolism and induces cell death. Beyond its pro-apoptotic and autophagy-inducing effects in breast cancer [14, 18], our findings expand upon this by showing that 8-Cl-Ado also induces ferroptosis, a non-apoptotic form of cell death driven by iron accumulation and lipid peroxidation. Exposure of breast cancer cell lines MCF-7 and MDA-MB-231 to 10 µM 8-Cl-Ado significantly increased Fe²⁺, MDA, and ROS levels while depleting GSH, consistent with a ferroptotic phenotype. Furthermore, both mRNA and protein levels of SLC7A11 were markedly reduced following treatment, suggesting that 8-Cl-Ado promotes ferroptosis via SLC7A11 inhibition. In vivo, administration of 8-Cl-Ado (100 mg/kg) significantly inhibited tumor growth in MCF-7 xenograft models, with marked tumor volume reduction observed by day 9. This rapid therapeutic response implies that 8-Cl-Ado targets critical cellular survival pathways. Immunohistochemical analysis of xenograft tumors confirmed reduced SLC7A11 expression in 8-Cl-Ado-treated tissues, in agreement with qRT-PCR and Western blot findings. These consistent results highlight the robustness and reliability of our data, and further support the role of 8-Cl-Ado in inducing ferroptosis via SLC7A11 downregulation.
To explore the upstream regulation of SLC7A11, we integrated miRNA array data with bioinformatic predictions from ENCORI and miRWalk databases, and identified miR-101-3p as a key candidate regulator. miR-101-3p is known to function as a tumor suppressor [35–37]. Dual-luciferase reporter assays confirmed that SLC7A11 is a direct target of miR-101-3p. Treatment with 8-Cl-Ado significantly increased miR-101-3p expression, leading to downregulation of SLC7A11 and induction of ferroptosis. Furthermore, overexpression of miR-101-3p in breast cancer cells increased Fe²⁺, MDA, and ROS levels while reducing GSH contents, mirroring the effects of 8-Cl-Ado treatment. These results indicate that miR-101-3p/SLC7A11 axis mediates ferroptosis downstream of 8-Cl-Ado.
We next investigated the mechanism by which 8-Cl-Ado regulates miR-101-3p. Prior studies have shown that 8-Cl-Ado inhibits ADAR1 expression by interfering with PU.1 binding to the ADAR1 promoter [18, 25]. ADAR1 is an RNA-editing enzyme that regulates both coding and non-coding RNAs, including miRNAs [30, 31]. In this study, we further confirmed that 8-Cl-Ado downregulates ADAR1. So, we hypothesized that ADAR1 negatively regulates miR-101-3p biogenesis. Indeed, knockdown of ADAR1 increased miR-101-3p levels, whereas ADAR1 overexpression reduced them. Given that mature miRNAs are processed from precursor transcripts, we further examined this regulation using FISH and RNA immunoprecipitation (RIP). FISH demonstrated altered intracellular distribution of pre-miR-101-2 and mature miR-101-3p following ADAR1 overexpression. RIP assays confirmed that ADAR1 binds to pre-miR-101-2, potentially interfering with its maturation into miR-101-3p. These findings suggest that ADAR1 suppresses miR-101-3p expression by binding to its precursor independent of its editing activity. It should be noted that since editing was not directly measured in this study, the term “editing-independent” is used here in the sense “strongly suggested by the ADAR1 ΔE/A rescue experiments”. Future work will elucidate the deep mechanism by which ADAR1 binding to pre-miR-101-2 impedes its processing into mature miR-101-3p, focusing on the identification of essential protein mediators.
Finally, functional rescue experiments confirmed that the ferroptosis-inducing effects of 8-Cl-Ado are mediated through ADAR1/miR-101-3p/SLC7A11 signaling. Modulating ADAR1 levels altered cellular ferroptosis markers in the presence of 8-Cl-Ado, further validating this regulatory axis. Our previous work demonstrated that ADAR1 is upregulated in breast cancer cell lines (including MDA-MB-231 (TNBC) and MCF-7) and breast cancer patients, where high ADAR1 expression correlates with poor overall survival and prognosis [26]. Therefore, inducing ferroptosis by targeting ADAR1 with 8-Cl-Ado represents a potential therapeutic strategy for breast cancer.

Conclusion
In summary, our study provides the first evidence that 8-Cl-Ado induces ferroptosis in breast cancer by targeting the ADAR1/miR-101-3p/SLC7A11 pathway independently of RNA-editing activity of ADAR1. These findings reveal a novel anti-tumor mechanism of 8-Cl-Ado and highlight its potential as a therapeutic agent for breast cancer. However, further investigations are warranted to clarify how ADAR1 binds to and suppresses the processing of pre-miR-101-2 into mature miR-101-3p. Future studies should also explore potential synergistic effects of combining 8-Cl-Ado with other ferroptosis-inducing agents or immunotherapies, and evaluate the translational potential of this approach in clinical settings. Together, our findings lay the groundwork for a novel ferroptosis-based therapeutic strategy against breast cancer.

Supplementary Information