Targeting the METTL3/PCNA axis with STM2457 overcomes cisplatin resistance in osteosarcoma

Introduction

Osteosarcoma (OS), the most common primary bone malignancy, predominantly affects adolescents and young adults, with cisplatin-based chemotherapy remaining a cornerstone of its clinical management (1,2). Despite initial therapeutic responses, the development of cisplatin resistance poses a major obstacle, leading to tumor recurrence and poor prognosis (3). Cisplatin resistance in OS is multifactorial, involving enhanced DNA repair, dysregulated apoptosis, and increased drug efflux (4). Among these mechanisms, proliferating cell nuclear antigen (PCNA), a pivotal regulator of DNA replication and repair, has emerged as a critical contributor to chemoresistance (5,6). PCNA overexpression is associated with aggressive tumor behavior and therapeutic failure in multiple cancers, including OS (7,8). As a sliding clamp that enhances DNA polymerase processivity, PCNA facilitates both translesion synthesis and homologous recombination repair, enabling tumor cells to bypass or repair cisplatin-induced DNA crosslinks, thereby directly conferring chemoresistance. However, the upstream molecular pathways driving PCNA dysregulation in cisplatin-resistant OS remain poorly understood, limiting the development of targeted strategies to restore chemosensitivity.

Recent advances in epitranscriptomics have highlighted the role of N6-methyladenosine (m6A) RNA modification in cancer progression and drug resistance (9,10). As the primary methyltransferase responsible for m6A deposition, methyltransferase-like 3 (METTL3) regulates messenger RNA (mRNA) stability, splicing, and translation, thereby influencing oncogenic pathways (11,12). In OS, METTL3 has been implicated in tumor growth and metastasis, yet its involvement in cisplatin resistance remains unexplored (11). Notably, METTL3-mediated m6A modification has been linked to the stabilization of DNA repair-related transcripts, suggesting a potential connection to PCNA regulation (13). This raises a critical hypothesis: METTL3 might orchestrate cisplatin resistance in OS by modulating PCNA expression via m6A-dependent mechanisms. Addressing this gap could unveil novel therapeutic targets to counteract chemoresistance. However, directly targeting PCNA has proven challenging due to its lack of enzymatic activity and essential role in normal cell division. Therefore, targeting its upstream regulators, such as the METTL3-m6A axis, presents a novel and potentially more viable strategy to indirectly suppress PCNA and overcome chemoresistance.

STM2457, a selective METTL3 inhibitor, has shown promise in suppressing m6A-dependent oncogenic signaling in hematological malignancies (14,15). However, its efficacy in solid tumors, particularly in the context of chemotherapy resistance, remains underexplored. Furthermore, no studies have investigated whether STM2457 can reverse cisplatin resistance by targeting PCNA or similar DNA repair regulators in OS. Given the unmet clinical need for adjuvant therapies to overcome chemoresistance and the lack of strategies targeting the epitranscriptomic regulation of DNA repair, evaluating STM2457’s potential to disrupt the METTL3-PCNA axis represents a strategically innovative approach.

In this study, we hypothesized that STM2457 mitigates cisplatin resistance in OS by inhibiting METTL3-mediated m6A methylation, thereby destabilizing PCNA mRNA and suppressing its pro-survival functions. Using cisplatin-resistant OS cell models, we aimed to: (I) determine the effects of STM2457 on cell proliferation, apoptosis, and migration; (II) elucidate the mechanistic link between METTL3 inhibition and PCNA downregulation; and (III) validate PCNA as a critical downstream effector of STM2457’s anti-chemoresistance activity. Our findings provide the first evidence that targeting METTL3 with STM2457 disrupts PCNA-driven survival pathways, offering a novel epigenetic strategy to resensitize refractory OS to cisplatin. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1429/rc).

Methods

Cell lines and culture conditions

Cisplatin-resistant OS cell lines MG-63/DDP and U2OS/DDP were purchased from the American Type Culture Collection (USA) and authenticated via short tandem repeat (profiling). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (HyClone, Logan, UT, USA) at 37 ℃ in a 5% CO₂ humidified incubator. To maintain cisplatin resistance, cells were routinely cultured with 2 µM cisplatin (Sigma-Aldrich, Louis, MO, USA). Cisplatin was removed 72 hours before experiments to avoid interference.

Reagents and antibodies

STM2457 (Cat# HY-138687, MedChemExpress, Monmouth Junction, NJ, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and stored at −80 ℃. Cisplatin (Sigma-Aldrich) was prepared in phosphate-buffered saline (PBS). Primary antibodies included anti-PCNA (1:1,000, Cat# 2586, Cell Signaling Technology, Danvers, MA, USA), anti-METTL3 (1:1,000, Cat# ab195352, Abcam, Cambridge, UK), anti-β-actin (1:5,000, Cat# 66009-1-Ig, Proteintech, Rosemont, IL, USA), and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, Proteintech).

Cell viability assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Cells (5×10³/well) were seeded in 96-well plates and treated with STM2457 (0–20 µM) alone or in combination with cisplatin (0–50 µM) for 48 hours. CCK-8 reagent (10 µL/well) was added, and absorbance at 450 nm was measured using a microplate reader (BioTek, Winooski, VT, USA). Half-maximal inhibitory concentration (IC₅₀) values were calculated via GraphPad Prism 9.0 using nonlinear regression.

Immunofluorescence staining

Cells were seeded at a density of 1×10⁶ cells per well and treated with STM2457 (10 µM), cisplatin (10 µM), or their combination for 48 hours. Following treatment, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and subsequently permeabilized with 0.1% Triton X-100 for 10 minutes. After washing, nuclear staining was performed using Hoechst 33342 (5 µg/mL; Cat# C1022, Beyotime Biotechnology, Shanghai, China) for 10 minutes. Immunostaining was then conducted through overnight incubation at 4 ℃ with an anti-cleaved Caspase-3 primary antibody (1:200 dilution; Cat# 9664, Cell Signaling Technology), anti-vimentin primary antibody (1:200 dilution; Cat# ab92547, Abcam) followed by a 1-hour incubation with Alexa Fluor 488-conjugated secondary antibody (1:500; Invitrogen, Carlsbad, CA, USA) at room temperature. For quantitative assessment, stained cells were visualized under a fluorescence microscope (Nikon Eclipse Ti, Nikon Corporation, Tokyo, Japan). The apoptotic rate or migration capacity was calculated as number of positive cells/total cells counted ×100% with ≥5 randomly selected fields analyzed per experimental group.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen). Complementary DNA (cDNA) was synthesized with the PrimeScript RT Master Mix (Takara, Kusatsu, Japan). qRT-PCR was performed using SYBR Green Premix (Roche, Basel, Switzerland) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as the internal gene. Relative mRNA levels were calculated using the 2^−ΔΔCt method. Primers for PCNA and GAPDH as follows:

PCNA: F, 5'-CCTGCTGGGATATTAGCTCCA-3'; R, 5'-GCACGTCCATTTCGTATTTGC-3';
GAPDH: F, 5'-GGAGCGAGATCCCTCCAAAAT-3'; R, 5'-GGCTGTTGTCATACTTCTCATGG-3'.

Western blot

Proteins were extracted using RIPA lysis buffer (Beyotime) and quantified via BCA assay (Thermo Fisher, Waltham, MA, USA). Equal amounts (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% non-fat milk, incubated with primary antibodies overnight at 4 ℃, and probed with HRP-conjugated secondary antibodies for 1 hour. Protein bands were visualized using enhanced chemiluminescence (ECL) reagent (Thermo Fisher) and quantified via Image Lab 6.0 (Bio-Rad, Hercules, CA, USA).

m6A RNA methylation quantification

Global m6A levels were measured using the EpiQuik m6A RNA Methylation Quantification Kit (Epigentek, Farmingdale, NY, USA). Total RNA (200 ng) was adsorbed to strip wells, incubated with capture and detection antibodies, and quantified colorimetrically at 450 nm. Data were normalized to input RNA.

PCNA knockdown and overexpression

siRNA targeting PCNA (si-PCNA: 5'-GCAUGAACUUCGACGCUAA-3') and scrambled siRNA (si-NC) were synthesized by GenePharma (Shanghai, China). For overexpression, full-length PCNA cDNA was cloned into the pcDNA3.1 vector (Invitrogen). Transfections were performed using Lipofectamine 3000 (Invitrogen) for 48 hours. Efficiency was validated by qRT-PCR and western blot.

Statistical analysis

Data are presented as mean ± standard deviation (SD) of three independent experiments. Differences between groups were analyzed by Student’s t-test or one-way analysis of variance (ANOVA) with Tukey’s post-hoc test using SPSS 26.0. Synergistic effects of drug combinations were assessed via the combination index (CI) using CompuSyn 1.0. The CI was calculated based on the Chou-Talalay method, which derives from the median-effect principle of the mass-action law. The general formula for CI is expressed as CI = (D)₁/(Dx)₁ + (D)₂/(Dx)₂, where (D)₁ and (D)₂ are the doses of drug 1 and drug 2 in combination that achieve a certain effect level (e.g., IC₅₀), and (Dx)₁ and (Dx)₂ are the doses of each drug alone required to produce the same effect. A CI <1, =1, or >1 indicates synergy, additive effect, or antagonism, respectively. A P value <0.05 was considered statistically significant.

Results

STM2457 suppresses proliferation of cisplatin-resistant OS cells and synergizes with cisplatin

The anti-proliferative efficacy of STM2457 against cisplatin-resistant OS cell lines (MG-63/DDP and U2OS/DDP) was assessed using a dose-response assay. Cells were treated with increasing concentrations of STM2457 (0–20 µM) for 48 hours.

STM2457 monotherapy significantly and concentration-dependently reduced cell viability in both resistant lines (Figure 1A). Critically, STM2457 treatment substantially sensitized the resistant cells, evidenced by marked reductions in their IC₅₀ values. Compared to untreated controls, the IC₅₀ of STM2457 decreased by 42.3% in MG-63/DDP cells (from 12.5±1.2 to 7.2±0.8 µM, **P<0.01) and by 44.7% in U2OS/DDP cells (from 14.1±1.4 to 7.8±0.9 µM, ^##P<0.01) (Figure 1A).

Figure 1 STM2457 inhibits proliferation and synergizes with cisplatin in cisplatin-resistant OS cells. (A) Dose-response curves of STM2457 (0–20 µM, 48 hours) on cell viability in MG-63/DDP and U2OS/DDP cells, as determined by CCK-8 assay. IC₅₀ values are shown as mean ± SD (n=3; **, P<0.01 vs. untreated control for MG-63/DD; ^##, P<0.01 vs. untreated control for U2OS/DDP). (B) CI analysis of STM2457 (10 µM) and cisplatin (10 µM) treatment for 48 hours. Synergistic effects (CI <1) are highlighted. Data represent mean ± SD (n=3; **, P<0.01; ***, P<0.001 vs. untreated control for MG-63/DDP; ^##, P<0.01; ^###, P<0.001 vs. untreated control for U2OS/DDP). CCK-8, Cell Counting Kit-8; CI, combination index; Cis, cisplatin; ns, not significant; IC₅₀, half-maximal inhibitory concentration; OS, osteosarcoma; SD, standard deviation.

To evaluate potential synergy with cisplatin, cells were co-treated with a fixed dose of STM2457 (10 µM, approximating the IC₅₀ range in these resistant lines) and cisplatin (10 µM). This combination treatment resulted in significantly enhanced growth inhibition beyond the effects of either agent alone (Figure 1B). Chou-Talalay CI analysis confirmed strong synergistic interaction in both cell lines (CI =0.62 for MG-63/DDP and CI =0.58 for U2OS/DDP; ***P<0.001 and ^###P<0.001 versus the respective cisplatin monotherapy group). CI value <1.0 indicates synergy.

STM2457 synergistically enhances cisplatin-induced apoptosis in resistant cells

Immunofluorescence analysis of cleaved Caspase-3 (a key executor protease of apoptosis) and nuclear morphology (Hoechst staining) was employed to assess apoptotic induction. Treatment with STM2457 (10 µM) alone elicited a moderate apoptotic response in cisplatin-resistant OS cells (MG-63/DDP: 13.1%±1.6%, *P<0.05; U2OS/DDP: 14.9%±1.7%, ^#P<0.05). Cisplatin monotherapy (10 µM) demonstrated limited pro-apoptotic efficacy in these resistant lines (MG-63/DDP: 8.7%±1.0%; U2OS/DDP: 9.9%±1.2%).

Strikingly, the combination of STM2457 and cisplatin induced a profound synergistic increase in apoptosis. The proportion of apoptotic cells surged to 29.3%±2.0% in MG-63/DDP cells and 32.1%±2.3% in U2OS/DDP cells (Figure 2). This represents a significant 3.4- and 3.2-fold enhancement compared to cisplatin monotherapy in MG-63/DDP (***P<0.001) and U2OS/DDP (^###P<0.001) cells, respectively. Apoptotic cells displayed characteristic morphological and biochemical hallmarks, including intense immunofluorescence signal for cleaved Caspase-3 (green) and condensed/fragmented nuclei (blue).

Figure 2 STM2457 enhances cisplatin-induced apoptosis in resistant OS cells. (A) Representative immunofluorescence images showing cleaved Caspase-3 (green), nuclear morphology (Hoechst, blue), and merged views. White arrows indicate apoptotic cells with nuclear fragmentation. Scale bar: 50 µm. (B) Quantification of apoptotic rates. Data are presented as mean ± SD (n=3; *, P<0.05; ***, P<0.001 vs. control for MG-63/DDP; ^#, P<0.05; ^###, P<0.001 vs. control for U2OS/DDP). DAPI, 4',6-diamidino-2-phenylindole; ns, not significant; OS, osteosarcoma; SD, standard deviation.

STM2457 suppresses the migratory phenotype of cisplatin-resistant OS cells

Immunofluorescence analysis revealed that STM2457 (10 µM) significantly inhibited the migratory capacity of cisplatin-resistant OS cells, as evidenced by reduced vimentin expression, a key marker of epithelial-mesenchymal transition associated with cell migration. Quantitative assessment demonstrated a substantial decrease in vimentin-positive cells upon STM2457 treatment. Specifically, in MG-63/DDP cells, the percentage of vimentin-positive cells was markedly reduced to 41.5%±4.1%, representing a significant 55.1% decrease compared to the control group (92.4%±3.2%, **P<0.01). Similarly, in U2OS/DDP cells, STM2457 treatment resulted in a reduction to 38.7%±3.8%, corresponding to a 57.4% decrease relative to controls (90.8%±3.5%, ^##P<0.01) (Figure 3).

Figure 3 STM2457 suppresses migration capacity of cisplatin-resistant OS cells. (A) Representative immunofluorescence images showing vimentin (red), nuclear morphology (Hoechst, blue), and merged views. White arrows indicate apoptotic cells with nuclear fragmentation. Scale bar: 50 µm. (B) Quantitative analysis of vimentin-positive cells. Data are mean ± SD (n=3; **, P<0.01; ***, P<0.001 vs. control for MG-63/DDP; ^##, P<0.01; ^###, P<0.001 vs. control for U2OS/DDP). DAPI, 4',6-diamidino-2-phenylindole; ns, not significant; OS, osteosarcoma; SD, standard deviation.

In contrast, cisplatin monotherapy (10 µM) exhibited only a limited suppressive effect on migration, with vimentin-positive cells remaining high (85.3%±3.5% for MG-63/DDP and 84.6%±3.2% for U2OS/DDP). Notably, the combination of STM2457 and cisplatin demonstrated a potent synergistic effect. The proportion of vimentin-positive cells was drastically reduced to 35.2%±3.8% in MG-63/DDP cells and 32.5%±3.5% in U2OS/DDP cells. This represents a significant 2.4- and 2.6-fold greater reduction compared to cisplatin treatment alone in MG-63/DDP (***P<0.001) and U2OS/DDP (^###P<0.001) cells, respectively.

STM2457 synergistically downregulates PCNA via METTL3-dependent m6A methylation

To elucidate the molecular mechanism underlying STM2457’s anti-proliferative and pro-apoptotic effects, we investigated its impact on the METTL3-m6A-PCNA regulatory axis. Crucially, this analysis encompassed STM2457 monotherapy, cisplatin monotherapy, and their combination. STM2457 monotherapy profoundly suppressed PCNA expression. Specifically, qRT-PCR demonstrated significant reductions in PCNA mRNA levels after STM2457 treatment (MG-63/DDP: 54.2%, *P<0.05; U2OS/DDP: 57.8%, ^#P<0.05) (Figure 4A). Cisplatin monotherapy alone exhibited only modest effects on PCNA mRNA levels (reduction <20%, P>0.05, Figure 4A) in these resistant lines. Strikingly, the combination of STM2457 (10 µM) and cisplatin (10 µM) induced significantly greater downregulation of PCNA mRNA in both cell lines than either agent alone (MG-63/DDP: 81.1%, ***P<0.001; U2OS/DDP: 84.4%, ^###P<0.001) (Figure 4A).

Figure 4 STM2457 downregulates PCNA via METTL3-dependent m6A methylation. (A) qRT-PCR analysis of PCNA mRNA levels in MG-63/DDP and U2OS/DDP cells treated with STM2457 (10 µM, 48 hours). GAPDH was used as a reference. Data are mean ± SD (n=3; *, P<0.05; ***, P<0.001 vs. control for MG-63/DDP; ^#, P<0.05; ^###, P<0.001 vs. control for U2OS/DDP). (B) Western blot analysis of PCNA protein expression. (C) Densitometric quantification of PCNA protein levels normalized to β-actin. Data are mean ± SD (n=3; *, P<0.05; ***, P<0.001 vs. control for MG-63/DDP; ^#, P<0.05; ^###, P<0.001 vs. control for U2OS/DDP). (D) Western blot analysis of METTL3 protein expression. (E) Densitometric quantification of METTL3 protein levels normalized to β-actin. Data are mean ± SD (n=3). (F) Global m6A RNA methylation levels measured by colorimetric assay in STM2457-treated cells (10 µM, 48 hours). Data are normalized to input RNA (mean ± SD, n=3; *, P<0.05 vs. control for MG-63/DDP; ^#, P<0.05 vs. control for U2OS/DDP). (G,H) PCNA mRNA stability assessed by qRT-PCR after actinomycin D (5 µg/mL) treatment in MG-63/DDP and U2OS/DDP cells under different treatments. Half-life (t_1/2) was calculated from decay curves (mean ± SD, n=3). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; METTL3, methyltransferase-like 3; mRNA, messenger RNA; ns, not significant; PCNA, proliferating cell nuclear antigen; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation.

Correspondingly, western blot analysis showed marked decreases in PCNA protein abundance after STM2457 treatment (MG-63/DDP: 48.6%, *P<0.05; U2OS/DDP: 52.3%, ^#P<0.05) (Figure 4B,4C). Cisplatin monotherapy alone exhibited only modest effects on PCNA protein expression (reduction <20%, P>0.05) in these resistant lines (Figure 4B,4C). In MG-63/DDP cells, combined treatment reduced PCNA protein levels by 72.1%±4.8% (vs. STM2457: 48.6%; vs. cisplatin: 15.3%, ***P<0.001) (Figure 4C). Similarly, in U2OS/DDP cells, PCNA protein was reduced by 75.6%±5.1% (vs. STM2457: 52.3%; vs. cisplatin: 18.7%, ^###P<0.001) (Figure 4C).

Notably, as shown in Figure 4D,4E, western blot analysis confirmed that STM2457 (10 µM) specifically inhibits METTL3 catalytic activity without altering its protein expression levels in both MG-63/DDP and U2OS/DDP cell lines, indicating that the observed reduction in m6A methylation is due to functional inhibition rather than a decrease in METTL3 protein. Consistent with METTL3 inhibition, global m6A quantification (via liquid chromatography-tandem mass spectrometry) revealed a significant reduction in total m6A levels upon STM2457 treatment (MG-63/DDP: 39.4% decrease, *P<0.05; U2OS/DDP: 41.7% decrease, ^#P<0.05) (Figure 4F). In contrast, cisplatin monotherapy (10 µM) exerted minimal effects on global m6A levels (<5% change, P>0.05). RNA stability assays confirmed that STM2457 dramatically accelerated PCNA mRNA decay, reducing its half-life in MG-63/DDP cells from 7.9±0.7 to 2.6±0.5 hours (**P<0.01) and in U2OS/DDP cells from 7.1±0.6 to 2.1±0.4 hours (^##P<0.01) (Figure 4G,4H). In contrast, cisplatin monotherapy (10 µM) exerted minimal effects on PCNA mRNA decay half-life, with t_1/2=7.3±0.6 hours in MG-63/DDP (<5% change vs. control, P>0.05) and t_1/2=6.8±0.5 hours in U2OS/DDP cells (<5% change vs. control, P>0.05). Strikingly, the potent suppression correlated with near-complete abrogation of PCNA mRNA stability (half-life <1 hour in both lines, P<0.001 vs. all monotherapies) (Figure 4G,4H).

These data establish that STM2457 exerts its anti-proliferative effects, at least in part, through METTL3-mediated suppression of m6A modification on PCNA mRNA, leading to its destabilization and reduced expression. The profound synergistic downregulation of PCNA by the STM2457-cisplatin combination provides a mechanistic basis for their enhanced anti-tumor efficacy observed in functional assays.

Functional validation: PCNA as a critical downstream effector of STM2457

To establish PCNA as a key functional mediator of STM2457’s activity, we employed siRNA-mediated PCNA knockdown (si-PCNA) in cisplatin-resistant OS cells. PCNA silencing significantly impaired cell viability, reducing it by 47.1%±3.8% in MG-63/DDP cells (**P<0.01) and 49.8%±4.2% in U2OS/DDP cells (^##P<0.01) compared to non-targeting siRNA controls (si-NC) (Figure 5A). This anti-proliferative effect closely mirrored that achieved by STM2457 (10 µM) monotherapy (MG-63/DDP: 48.2%±4.1%, **P<0.01 vs. si-NC; U2OS/DDP: 51.7%±4.5% reduction, ^##P<0.01 vs. si-NC), strongly suggesting PCNA downregulation is a primary mechanism underlying STM2457 efficacy. Conversely, forced overexpression of PCNA (using pcDNA3.1-PCNA vector) significantly attenuated the cellular responses to STM2457. PCNA overexpression partially reversed STM2457-mediated suppression of cell viability (Figure 5A, P>0.05 vs. si-NC). In STM2457-treated cells, viability was increased by 1.8-fold ±0.2 (**P<0.01) in MG-63/DDP and 1.7-fold ±0.2 (^##P<0.01) in U2OS/DDP compared to STM2457-treated cells transfected with empty vector (Figure 5A).

Figure 5 PCNA is a critical functional mediator of STM2457’s anti-tumor effects in cisplatin-resistant osteosarcoma cells. (A) Cell viability after siRNA-mediated PCNA knockdown (si-PCNA) or STM2457 (10 µM) treatment in MG-63/DDP and U2OS/DDP cells (CCK-8 assay; mean ± SD, n=3; **, P<0.01 vs. si-NC for MG-63/DDP; ^##, P<0.01 vs. si-NC for U2OS/DDP). (B,C) Immunofluorescence images (B) and quantification (C) of apoptosis (cleaved Caspase-3 staining): PCNA overexpression decreased STM2457-induced apoptosis. Data are mean ± SD (n=3; **, P<0.01 vs. vector + NC for MG-63/DDP; ^##, P<0.01 vs. vector + NC for U2OS/DDP). (D,E) Representative immunofluorescence images (D) and quantification (E) of migration capacity (vimentin-positive cells). PCNA overexpression restored STM2457-inhibited migration. Scale bar: 50 µm. Data are mean ± SD (n=3; *, P<0.05 vs. vector + NC for MG-63/DDP; ^#, P<0.05 vs. vector + NC for U2OS/DDP). (F) Cisplatin resistance restoration: PCNA overexpression increased cisplatin IC₅₀, diminishing STM2457’s chemosensitization effect. Data are mean ± SD (n=3; ***, P<0.001 vs. vector + Cis for MG-63/DDP; ^###, P<0.001 vs. vector + Cis for U2OS/DDP). White arrows indicate apoptotic cells with nuclear fragmentation. CCK-8, Cell Counting Kit-8; Cis, cisplatin; DAPI, 4',6-diamidino-2-phenylindole; IC₅₀, half-maximal inhibitory concentration; NC, negative control; ns, not significant; PCNA, proliferating cell nuclear antigen; SD, standard deviation; si-NC, scrambled negative control siRNA.

Critically, PCNA overexpression conferred significant protection against STM2457-induced apoptosis (Figure 5B,5C). The proportion of apoptotic cells (cleaved Caspase-3+) was reduced by 58.3%±5.1% in MG-63/DDP (**P<0.01) and 61.7%±5.5% in U2OS/DDP (^##P<0.01) compared to STM2457-treated, empty vector controls (Figure 5B,5C). Similarly, PCNA overexpression significantly restored the migratory capacity suppressed by STM2457 (Figure 5D,5E). Vimentin-positive cells increased by 1.7-fold ±0.2 in both MG-63/DDP (*P<0.05) and U2OS/DDP (^#P<0.05) lines compared to STM2457-treated, empty vector controls (Figure 5D,5E).

Importantly, PCNA overexpression not only counteracted STM2457’s effects but also partially re-established the cisplatin-resistant phenotype. In cells overexpressing PCNA, the sensitization effect of STM2457 on cisplatin cytotoxicity was significantly diminished (Figure 5F). The IC₅₀ of cisplatin in STM2457-treated and PCNA-overexpressing groups is higher than in STM2457-treated cells with empty vector (MG-63/DDP: 1.9-fold higher, *P<0.05; U2OS/DDP: 2.1-fold higher, ^#P<0.05) (Figure 5F).

These gain- and loss-of-function experiments definitively establish PCNA as a critical functional target downstream of STM2457-mediated METTL3/m6A inhibition. The ability of PCNA overexpression to reverse STM2457’s anti-proliferative, anti-migratory, and pro-apoptotic effects, and to partially restore cisplatin resistance, underscores its pivotal role in mediating both the intrinsic anti-tumor activity of STM2457 and its synergy with cisplatin in overcoming chemoresistance.

Discussion

The emergence of cisplatin resistance in OS remains a formidable barrier to effective treatment, underscoring the urgent need to identify actionable targets that can restore chemosensitivity. Our study unveils a previously unrecognized role of the METTL3 inhibitor STM2457 in counteracting cisplatin resistance through the suppression of PCNA, a master regulator of DNA repair and tumor progression. By integrating phenotypic analyses with mechanistic exploration, we demonstrate that STM2457 not only inhibits proliferation and migration but also synergizes with cisplatin to induce apoptosis in resistant OS cells. These effects are mechanistically rooted in the METTL3-m6A-PCNA axis, where STM2457 reduces m6A-dependent stabilization of PCNA mRNA, thereby destabilizing a critical survival pathway. This work expands the therapeutic potential of METTL3 inhibition beyond hematological malignancies, positioning STM2457 as a promising epigenetic adjuvant for chemoresistant solid tumors.

Our findings align with emerging evidence implicating METTL3 in chemoresistance but diverge in their translational focus. For instance, METTL3 has been shown to stabilize DNA repair genes in non-small cell lung cancer (16), conferring resistance to platinum drugs (17), and its inhibition enhances chemosensitivity in leukemia (14). However, prior studies have not addressed whether METTL3-driven m6A modification regulates PCNA in OS or whether such regulation can be therapeutically targeted (11). Here, we establish PCNA as a direct downstream effector of METTL3 in cisplatin-resistant OS, bridging the gap between epitranscriptomic dysregulation and chemoresistance. Importantly, our observation that PCNA knockdown phenocopies STM2457’s effects, while its overexpression partially rescues them, provides functional validation of this axis, a critical advance over correlative studies in the field.

The innovation of this study lies in its dual focus on METTL3’s enzymatic activity and its therapeutic inhibition. Unlike genetic approaches (e.g., METTL3 siRNA), which may confound interpretation due to off-target effects, STM2457 specifically targets METTL3’s methyltransferase domain, offering a pharmacologically tractable strategy. This distinction is crucial, as our data confirm that STM2457 reduces global m6A levels without altering METTL3 protein expression rather than protein degradation, suggesting that catalytic inhibition drives its anti-tumor effects. Furthermore, the synergy between STM2457 and cisplatin highlights the potential of combining epigenetic modulators with conventional chemotherapy to overcome resistance, a concept gaining traction in oncology but underexplored in OS.

There are limitations in this study. First, the reliance on cell line models, while informative, does not fully recapitulate the tumor microenvironment or interpatient heterogeneity. Future work should validate these findings in patient-derived xenografts or clinical samples to assess translational relevance. Second, while we focused on PCNA as a key target, METTL3 likely regulates additional genes contributing to cisplatin resistance. For example, m6A modification has been linked to drug efflux pumps (e.g., ATP-binding cassette transporters) and anti-apoptotic proteins (e.g., B-cell lymphoma 2) in other cancers, suggesting that STM2457’s efficacy may involve broader transcriptomic remodeling (18,19). Third, the long-term safety and pharmacokinetic profile of STM2457 in vivo remain to be characterized, particularly in combination with cisplatin, which may exhibit overlapping toxicities. Notwithstanding these limitations, our findings highlight the promising clinical translational potential of targeting METTL3 in chemoresistant OS. The potent synergy between STM2457 and cisplatin in vitro provides a strong rationale for future in vivo studies using patient-derived xenograft models to validate efficacy in a more physiologically relevant context. Prior studies of STM2457 in leukemia models have reported manageable toxicity profiles, suggesting a potentially favorable therapeutic window; however, its safety in combination with cisplatin in solid tumors warrants careful investigation. To translate this strategy to the clinic, optimizing combination dosing schedules will be crucial to maximize efficacy while minimizing potential overlapping toxicities, such as myelosuppression or nephrotoxicity.

Looking ahead, several directions merit exploration. Investigating whether STM2457 resensitizes resistant tumors to other DNA-damaging agents (e.g., doxorubicin) could broaden its clinical utility. Additionally, profiling the m6A epitranscriptome in STM2457-treated cells may uncover novel resistance-related targets beyond PCNA. Finally, the development of predictive biomarkers will be essential for patient stratification in potential clinical trials. High baseline expression of METTL3 or PCNA in tumor tissues could serve as a companion diagnostic to identify OS patients most likely to benefit from STM2457-containing combination regimens. This biomarker-driven approach would enhance the clinical applicability and success rate of this epigenetic strategy, paving the way for more personalized and effective treatment of chemoresistant OS.

Conclusions

In summary, this study demonstrates that the METTL3 inhibitor STM2457 effectively overcomes cisplatin resistance in OS by disrupting the METTL3-m6A-PCNA axis. Our findings reveal that STM2457 suppresses global m6A methylation, destabilizes PCNA mRNA, and reduces PCNA protein expression, thereby inhibiting tumor cell proliferation and migration while promoting apoptosis. The synergistic effect observed between STM2457 and cisplatin provides a compelling rationale for combining epigenetic targeted therapy with conventional chemotherapy. Targeting METTL3 represents a promising novel strategy to reverse chemoresistance in OS, warranting further investigation in preclinical and clinical settings.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1429/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1429/dss

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