lncRNA Deficiency Drives Cisplatin Resistance via NR2F1-Mediated TGFB1/NF-κB Signaling Axis in NSCLC.

1. Introduction
Non-small cell lung cancer (NSCLC) is the most prevalent histological subtype of lung cancer, accounting for approximately 85% of all cases and representing a leading cause of global cancer-related mortality [1]. For patients with advanced or post-operative disease, platinum-based chemotherapy, predominantly with cisplatin, remains a cornerstone of first-line treatment [2,3]. However, the clinical efficacy of cisplatin is severely limited by the frequent development of intrinsic or acquired drug resistance, which constitutes a major obstacle to achieving durable therapeutic responses and improving long-term survival [4,5]. Consequently, elucidating the underlying molecular mechanisms of cisplatin resistance is imperative for developing novel strategies to overcome this barrier and improve outcomes for NSCLC patients.
Long non-coding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides that lack protein-coding capacity. Accumulating evidence indicates their pivotal involvement in tumor biology, where they can act as drivers or suppressors of cancer initiation, progression, and metastasis [6]. Notably, their dysregulation is also increasingly linked to the development of chemotherapy resistance [7]. NR2F1-AS1 (nuclear receptor subfamily 2 group F member 1 antisense RNA 1, hereafter referred to as NAS1), a recently identified lncRNA located on chromosome 5q15, has been documented in a wide array of malignancies with context-dependent regulatory functions [8]. Intriguingly, the adjacent gene NR2F1 encodes a transcription factor reported to be translationally regulated by NAS1 and implicated in processes such as tumor dormancy and drug resistance [9,10]. However, the precise mechanism through which NAS1 modulates cisplatin resistance via NR2F1 in NSCLC has not yet been fully characterized. Therefore, this study aims to determine whether the NAS1-NR2F1 axis is involved in cisplatin resistance in NSCLC and to explore the molecular mechanisms by which its downstream factors exert their effects.
In this study, we identify that both NAS1 and NR2F1 are significantly downregulated in cisplatin-resistant NSCLC cell lines. Functional experiments confirm that knockdown of either gene increases cisplatin resistance, establishing their negative correlation with the cisplatin-resistant phenotype. Through rescue investigations, we demonstrate that NR2F1 is a necessary downstream target through which NAS1 exerts its effect. Furthermore, by integrating transcriptomic analysis and experimental validation, we reveal that downregulation of the NAS1-NR2F1 axis converges on the transcriptional derepression of TGFB1, leading to subsequent activation of the NF-κB signaling pathway to drive cisplatin resistance. Collectively, our findings delineate a NAS1/NR2F1/TGFB1/NF-κB regulatory axis and suggest that targeting this pathway could represent a promising therapeutic strategy for overcoming cisplatin resistance in NSCLC.

2. Materials and Methods

2.1. Cell Culture
The human lung adenocarcinoma cell line PC9 (CellCook, CC0204, Guangzhou, China) and its cisplatin-resistant derivative PC9/CDDPr (CellCook, DR0204-DDP); the large cell lung cancer cell line H460 and its cisplatin-resistant derivative H460/CDDPr (MingJing Biology, M-C7062, Shanghai, China); as well as the lung squamous carcinoma cell line H226 (Cell Bank of Chinese Academy of Sciences, SCSP-5073, Shanghai, China) and its cisplatin-resistant counterpart H226/CDDPr were used in this study. Generated in-house by concentration-gradient intermittent induction: H226 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. H226 cisplatin-resistant cells were established in-house by stepwise intermittent exposure to increasing concentrations of cisplatin over 6 months until a stable resistant phenotype was achieved. Resistant cells were maintained in medium containing 1 μM cisplatin and cultured in drug-free medium for 1 week before experiments. All cell lines were authenticated by short tandem repeat profiling within the past 3 years and routinely confirmed to be free of mycoplasma contamination. Cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2. All cells were cultured in RPMI-1640 medium (BI, 01-100-1ACS, Kibbutz Beit Haemek, Israel) supplemented with 10% FBS (VivaCell, C04001-500, Shanghai, China) and 1% penicillin/streptomycin (Life Technologies, 15140163, Shanghai, China). To maintain the resistant phenotype, H226/CDDPr, H460/CDDPr and PC9/CDDPr cells were routinely cultured with 1 µM, 0.83 µM and 6.6 µM cisplatin, respectively. Cells were switched to drug-free medium for one week prior to experiments. All cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2.

2.2. RNA-Seq Data Analysis
For transcriptomic profiling of cisplatin-resistant cell lines, total RNA was extracted from cells at 70–80% confluence using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Libraries were prepared and sequenced on an Illumina NovaSeq platform (Novogene, Beijing, China). Raw transcriptome sequencing data were first subjected to quality control using FastQC (version 0.12.1) and summarized with MultiQC [11] (version 1.33). Low-quality bases and residual adapter sequences were then removed using TrimGalore (https://github.com/FelixKrueger/TrimGalore, accessed on 7 September 2016). The cleaned reads were aligned to the human reference genome (GRCh38, Ensembl release) using Hisat2 (version 2.2.1) [12]. Gene-level read counts were quantified with FeatureCounts [13]. Differential expression analysis was performed using DESeq2 (version 1.42.1) [14]. Genes with an absolute log2 fold change (|log2FC|) > 1 and a p-value < 0.05 were considered significantly differentially expressed.
For the external dataset GSE233167 (gastric cancer cells with NR2F1 knockdown or overexpression), FPKM values were downloaded from the GEO database. Genes with FPKM fold-change > 1.3 or <1/1.3 were defined as upregulated or downregulated, respectively. Heatmaps were generated for genes with |Z-score (OE_NC − KD_NC)| < 1.

2.3. TCGA Data Analysis
The RNA expression profiles of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) cohorts were obtained from The Cancer Genome Atlas (TCGA) database. Primary non-small cell lung cancer (NSCLC) tumor tissues and corresponding normal lung tissues were included in the analysis. The RNA expression level of NR2F1-AS1 was extracted from the TCGA expression datasets. Differences in NR2F1-AS1 expression between NSCLC and normal tissues were visualized using the ggplot2 package in R (version 4.0.1). The Wilcoxon rank-sum test was applied to evaluate the statistical significance of differences in NR2F1-AS1 RNA expression between the two groups.

2.4. Plasmid Construction and Transfection
To construct the shNR2F1 plasmid, a double-stranded oligonucleotide encoding the NR2F1-targeting shRNA (5′-CTCTTCTTCGTCCGTTTGGTA-3′) or a control shRNA (shScramble: 5′-AACAGTCGCGTTTGCGACTGG-3′) was cloned into the pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro vector. Correct insertion was verified by Sanger sequencing.
For NR2F1 overexpression, the full-length human NR2F1 coding sequence (NM_005654.6) was amplified by PCR with flanking HindIII and BamHI sites and inserted into the pcDNA3.1 vector to generate pcDNA3.1-Myc-NR2F1. Empty pcDNA3.1 was used as a negative control. Primer sequences are listed in Supplementary Table S1.
Cells were seeded in 3.5 cm dishes 24 h before transfection to reach 50–70% confluence. the pcDNA3.1 empty vector (negative control) or the pcDNA3.1-Myc-NR2F1 expression plasmid was delivered using Lipofectamine™ 3000 (Invitrogen, L3000015). Medium was replaced 4–6 h post-transfection, and subsequent experiments were conducted 24 h after transfection. Transfection efficiency was assessed 48 h after the transfection.

2.5. Construction of Knockdown Cell Lines
For stable NAS1 knockdown, lentiviral particles carrying two independent shRNAs (shNAS1-1: 5′-GACACTGATATAACTGTAGAT-3′; shNAS1-2: 5′-GCTGCATCCTTATGGTAGCTA-3′) or a control shRNA (shNC: 5′-TTCTCCGAACGTGTCACGT-3′) were obtained from GenePharma (Shanghai, China). Cells were co-infected with both shNAS1 lentiviruses or infected with shNC lentiviruses at 40–50% confluence. After 24 h, the viral supernatant was removed. The cells were then cultured in complete culture medium without antibiotics for an additional 24 h. Subsequently, the cells were maintained in complete culture medium for 2 days, followed by puromycin selection (PC9: 1 µg/mL, H226: 0.5 µg/mL) was applied for 7 days to establish stable pools.
For NR2F1 knockdown, lentivirus was produced by transfecting HEK-293T cells with packaging and shNR2F1 plasmids using Exfect Transfection Reagent (Vazyme, T101-02, Nanjing, China). Supernatants were collected at 24 h and 48 h, filtered (0.45 µm), and used to infect target cells at 40–50% confluence. After 24 h, complete medium was applied, and stable populations were selected with puromycin for 7 days. Knockdown efficiency was confirmed by qRT-PCR and Western blot.

2.6. RNA Isolation and Quantitative Real-Time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen, 15596018). Reverse transcription was performed using 1 µg RNA with HiScript III RT SuperMix (Vazyme, R323-01). qPCR was carried out with ChamQ SYBR Master Mix (Vazyme, Q331-02) on a QuantStudio 3 system (Applied Biosystems, Foster City, CA, USA). Relative expression was calculated by the 2−ΔΔCT method using β-actin as the internal control. All primers were synthesized by Tsingke and are listed in Supplementary Table S1.

2.7. Western Blot (WB)
Cells were lysed in SDS-PAGE sample buffer and heated at 100 °C for 10 min. Proteins were separated on 10% SDS-PAGE gels and transferred onto NC membranes (Sangon Biotech, F619512, Shanghai, China). Membranes were blocked with 5% non-fat milk in TBST, then incubated overnight at 4 °C with the following primary antibodies: COUP-TF1 (ab181137, Abcam, 1:1000, Cambridge, MA, USA), GAPDH (sc-365062, Santa Cruz, 1:5000, Santa Cruz, CA, USA), p-P65 (3033, CST, 1:1000, Danvers, MA, USA), P65 (8242, CST, 1:1000). After washing, membranes were incubated with HRP-conjugated anti-rabbit (SA00001-2, Proteintech, 1:5000, Wuhan, China) or anti-mouse (SA00001-1, Proteintech, 1:5000) secondary antibodies for 1 h at room temperature. Signals were detected with a ChemiDoc system (Tanon, Shanghai, China) and quantified using ImageJ (version 1.54g). All experiments were performed in triplicate.

2.8. Chemosensitivity Assay
Cell viability was assessed using the CCK-8 kit (Vazyme, A311-02). Cells were seeded in 96-well plates (3000 cells/well) and treated with a series of cisplatin concentrations (two-fold dilutions from 0 to 256 µM) for 48 h. Then, 10 µL of CCK-8 reagent was added per well, incubated for 1 h, and absorbance was measured at 450 nm. The half-maximal inhibitory concentration (IC50) was calculated by fitting a dose–response curve in GraphPad Prism 10 using the equation inhibitor vs. normalized response-variable slope.

2.9. Cell Migration Assay
Migration was evaluated using Transwell chambers (8 µm pore, LabSelect, 14341, Beijing, China). Cells were resuspended in medium with 1% FBS and seeded into the upper chamber (6 × 105 cells/mL, 100 µL/well). The lower chamber contained medium with 10% FBS. Cisplatin (0, 4, or 8 µM) was added to both chambers. After 24 h, cells on the upper surface were removed, and migrated cells on the lower surface were fixed with 4% paraformaldehyde (Biosharp, 23355142, Beijing, China), stained with 0.1% crystal violet (Beyotime Biotechnology, C0121, Shanghai, China), imaged, and counted.

2.10. Colony Formation Assay
Cells were plated in 6-well plates at 700 cells/well. After adhesion, cisplatin (0, 0.5, or 1 µM) was added. Cells were cultured for 7–10 days, fixed with 4% paraformaldehyde (Biosharp, 23355142), stained with 0.1% crystal violet (Beyotime Biotechnology, C0121), and colony areas were quantified.

2.11. Enrichment Analysis
Gene Ontology and pathway enrichment analysis was performed using the clusterProfiler (v4.12.6) R package.

2.12. Statistical Analysis
All data are derived from at least three independent biological replicates. Unless otherwise specified, differences between two groups were assessed using unpaired two-tailed Student’s t-test. Data are presented as mean ± SEM. Significance levels are denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001. Analyses were performed in GraphPad Prism 10.

3. Results

3.1. NAS1 Is Downregulated in Multiple Cisplatin-Resistant NSCLC Cells
To investigate the role of lncRNAs in cisplatin resistance of NSCLC, we performed transcriptome-wide RNA sequencing to identify differentially expressed lncRNA between three distinct cisplatin-resistant NSCLC cell lines (H226/CDDPr, H460/CDDPr, PC9/CDDPr) and their corresponding parental cells (Figure 1A–C). Widespread dysregulation of lncRNA expression was observed across all resistant cell lines. Notably, H226-resistant cells exhibited thousands of differentially expressed genes, whereas PC9-resistant cells showed a comparatively limited transcriptional response. Analysis revealed two consistently downregulated and eight consistently upregulated lncRNAs across all three resistant cell lines (Figure 1D and Figure S1A). Subsequent validation by quantitative PCR (qPCR) confirmed that only NAS1 was significantly downregulated in all three cisplatin-resistant NSCLC models (Figure 1E and Figure S1B–E). Therefore, we further analyzed The Cancer Genome Atlas (TCGA) NSCLC cohorts and found that NAS1 expression was consistently low in NSCLC (Figure 1F). These findings suggest that NAS1 may play an important role in regulating cisplatin resistance in NSCLC.

3.2. NAS1 Knockdown Increases Cisplatin Resistance in NSCLC Cells
To validate the negative correlation between NAS1 expression and cisplatin resistance, we knocked down NAS1 in H226 and PC9 cell lines by co-transfecting two independent shRNAs targeting different regions of the NAS1 transcript (Figure 2A,B). Evaluation of the effect of NAS1 knockdown on cisplatin sensitivity showed that loss of NAS1 led to a roughly 2-fold elevation in the IC50 of cisplatin in both cell lines examined (Figure 2C–F). Moreover, NAS1 depletion markedly attenuated the inhibitory effects of cisplatin on colony formation and cell migration (Figure 2G–J). Taken together, these findings implicate that downregulation of NAS1 promotes cisplatin resistance in NSCLC.

3.3. The NAS1-Translational Regulated Gene NR2F1 Confers Cisplatin Resistance to NSCLC Cells upon Its Downregulation
To elucidate the molecular mechanism by which NAS1 regulates cisplatin resistance, we focused on NR2F1, a gene located adjacent to NAS1 that encodes a transcription factor previously reported to be translationally regulated by NAS1 and implicated in drug resistance [9,10]. To assess whether NAS1 mediates cisplatin resistance through NR2F1, we first confirmed their regulatory relationship. Consistent with prior studies, NAS1 knockdown reduced NR2F1 protein levels without affecting its mRNA expression (Figure 3A,B). Notably, both NR2F1 mRNA and NR2F1 protein levels were downregulated in cisplatin-resistant cells (Figure 3C,D), suggesting that reduced NR2F1 expression may contribute to cisplatin resistance. We then knocked down NR2F1 (Figure 3E,F) and evaluated its effect on drug sensitivity. Similar to NAS1 knockdown, NR2F1 depletion significantly increased the IC50 of cisplatin (Figure 3G,H) and attenuated cisplatin-induced suppression of colony formation and cell migration (Figure 3I–L). These findings indicate that NR2F1 downregulation promotes cisplatin resistance and that NR2F1 appears to act as a downstream target of NAS1 in mediating the resistant phenotype.

3.4. Overexpression of NR2F1 Alleviates Cisplatin Resistance Caused by NAS1 Knockdown
To further verify whether NR2F1 is required for NAS1-mediated cisplatin resistance, we performed a rescue experiment by overexpressing NR2F1 in two cisplatin-resistant cell lines (in which both NAS1 and NR2F1 are downregulated) and evaluated its impact on drug sensitivity (Figure 4A,B). The results showed that NR2F1 overexpression significantly reduced the IC50 of cisplatin in both resistant cell lines (Figure 4C–F). In H226-CDDPr cells, the IC50 decreased from approximately 13.91 μM to 7.49 μM, representing a nearly 2-fold reduction. In PC9-CDDPr cells, a more pronounced decrease was observed, with the IC50 dropping from 16.92 μM to 5.42 μM. These findings indicate that restoring NR2F1 expression effectively sensitizes cisplatin-resistant cells to treatment.
Moreover, restoring NR2F1 expression effectively rescued the cisplatin resistance induced by NAS1 knockdown (Figure 4G–L). In H226 cells, NR2F1 restoration lowered the IC50 from 14.1 μM to 4.63 μM, a reduction of approximately 67.16%. In PC9 cells, the IC50 decreased even more sharply, from 4.04 μM to 1.19 μM, corresponding to a 70.54% reduction. Collectively, these findings demonstrate that NR2F1 is a functional downstream target of NAS1 and is necessary for mediating cisplatin resistance in NSCLC.

3.5. NR2F1 Regulates Cisplatin Resistance in NSCLC Through the Transcriptional Repression of TGFB1
To elucidate the downstream molecular mechanisms by which the NAS1-NR2F1 axis regulates cisplatin resistance, we analyzed RNA-seq datasets (GSE233167) from gastric cancer cells following NR2F1 knockdown or overexpression. Given that the transcription factor NR2F1 has been reported to act as a transcriptional repressor of ΔNp63 [9], we focused on genes negatively regulated by NR2F1 (i.e., upregulated upon NR2F1 knockdown and downregulated upon NR2F1 overexpression) (Figure 5A). GO enrichment analysis revealed that these NR2F1-repressed genes were enriched in multiple drug resistance-associated signaling pathways (Figure 5B), which were similarly enriched among genes upregulated in cisplatin-resistant cells (Figure 5C). Among these pathways, NF-κB signaling, PI3K/AKT signaling, and the ERK1/ERK2 cascade have previously been linked to cisplatin resistance upon activation [4,15]. Notably, transforming growth factor-β1 (TGFβ1, TGFB1) was the only NR2F1-repressed gene involved in all three pathways (Figure 5D) and has been reported to promote drug resistance in various cancers [16,17,18]. These findings suggested that TGFB1 may be a key downstream target of the NAS1-NR2F1 axis. Subsequent qPCR validation confirmed that TGFB1 expression was upregulated in cisplatin-resistant NSCLC models as well as in NAS1- and NR2F1-knockdown cells (Figure 5E–G). Western blot analysis further confirmed the protein-level changes in NR2F1 and TGFB1 in these models. Consistent with the transcriptional results, cisplatin-resistant, NAS1-knockdown, and NR2F1-knockdown H226 and PC9 cells showed reduced NR2F1 protein expression accompanied by increased TGFB1 protein levels (Figure 5H–J). Together, these results demonstrate that TGFB1 acts as a downstream effector of the NAS1-NR2F1 axis and confers cisplatin resistance through the regulation of multiple signaling pathways.

3.6. Activation of the NF-κB Pathway by the Downregulated NAS1-NR2F1 Axis Drives Cisplatin Resistance
NF-κB hyperactivation is a known driver of chemoresistance, often through the upregulation of anti-apoptotic genes [15,19,20]. Furthermore, TGFB1 has been shown to activate NF-κB in other cancer contexts to promote a drug-resistant phenotype [18]. To test this link in our model, we examined the NF-κB activity. Immunoblot analysis revealed a consistent increase in the levels of phosphorylated p65 (p-p65), a marker of NF-κB activation, in both H226 and PC9 cisplatin-resistant cells (Figure 1E and Figure 6A,B). Notably, this elevation in p-p65 was inversely correlated with the downregulated expression of both NAS1 and NR2F1 in these resistant cells (Figure 1E and Figure 6A,B). We then performed loss-of-function experiments. Strikingly, the specific knockdown of either NAS1 or NR2F1 in parental NSCLC cells was sufficient to recapitulate the resistance-associated phenotype, leading to a marked increase in p-p65 levels (Figure 6C–F) and subsequent NF-κB transcriptional activity. To further determine whether NF-κB activation functionally contributes to cisplatin resistance, we treated cisplatin-resistant H226/CDDPr and PC9/CDDPr cells with the NF-κB inhibitor DHMEQ and assessed cisplatin sensitivity. DHMEQ treatment reduced the IC50 of cisplatin in both resistant cell lines in a dose-dependent manner, indicating that inhibition of NF-κB partially restored cisplatin sensitivity (Figure 6G,H). These results provide functional evidence that NF-κB activation contributes to the maintenance of the cisplatin-resistant phenotype in NSCLC cells.
Collectively, these findings delineate a comprehensive signaling cascade underlying cisplatin resistance in NSCLC. We proposed a model in which downregulation of NAS1 impairs the translation of NR2F1. The subsequent loss of NR2F1 relieves its transcriptional repression of TGFB1, leading to increased TGFB1 expression. Upregulated TGFB1 subsequently activates the downstream NF-κB pathway, ultimately driving the acquisition and maintenance of the cisplatin-resistant phenotype.

4. Discussion
The development of cisplatin resistance remains a major obstacle in the treatment of NSCLC. It is crucial to improve chemosensitivity and prevent or bypass chemoresistance to enhance the prognosis of NSCLC patients [21]. Therefore, developing novel potential targets related to chemotherapeutic treatment response rates, as well as revealing the underlying mechanisms, is essential for optimizing clinical chemotherapeutical schemes and the treatment of NSCLC. Emerging evidence has highlighted the involvement of NAS1, including NSCLC, in tumor development [8], but the specific roles and mechanisms of NAS1 in cisplatin resistance are poorly understood. By analyzing our RNA sequencing data, we identified lncRNA NAS1 as the most considerably down-regulated lncRNA in three cisplatin-resistant NSCLC cell lines compared to their corresponding parental cells (Figure 1). Knockdown of NAS1 further confirmed the negative relationship of NAS1 and cisplatin resistance of NSCLC (Figure 2). While numerous studies have established that NAS1 mainly acts as an oncogene across multiple cancer types, tumor-suppressive properties have been documented in specific malignancies, indicating context-dependent regulatory functions that differ among cancer types [8]. In the present study, TCGA database analysis demonstrated significant downregulation of NAS1 in NSCLC tumors (Figure 1F). However, phenotypic assays following NAS1 knockdown revealed that although NAS1 depletion significantly increased cellular chemoresistance, it concomitantly impaired cell migratory capacity (Figure 2I,J). Comparable observations were made in NR2F1 knockdown experiments (Figure 3K,L), underscoring the multifaceted role of the NAS1-NR2F1 axis in NSCLC. This functional complexity may be attributed to the regulatory effects of the NAS1-NR2F1 axis on cellular dormancy. It has been previously reported that NAS1 is upregulated in dormant mesenchymal-like breast cancer cells, where it promotes tumor dissemination through translational regulation of NR2F1, albeit at the expense of proliferative capacity [9]. Nevertheless, the specific role of the NAS1-NR2F1 axis in modulating cancer phenotypes in NSCLC warrants further investigation.
LncRNAs can modulate gene expression through diverse mechanisms, including functioning as competing endogenous RNAs (ceRNAs) or miRNA sponges, regulating RNA-binding proteins (RBPs), and transcription-dependent activation or repression of neighboring genes [22]. NR2F1, located adjacent to NAS1, has been previously reported to undergo translational regulation by NAS1, which binds NR2F1 mRNA and recruits the RBP PTBP1 to facilitate internal ribosome entry site (IRES)-mediated translation [9]. Our study confirmed this translational regulatory relationship between NAS1 and NR2F1 (Figure 3A). Whether post-translational regulation or altered protein stability contributes to NR2F1 downregulation remains an open question that will be important to address in future studies. The NAS1-NR2F1 axis has been implicated in cellular dormancy regulation, though the underlying mechanisms vary across different cellular contexts: it promotes dormancy in breast cancer cells but drives the transition from dormancy to proliferation in prostate cancer [9,23]. In contrast, we demonstrated that diminished expression of the NAS1-NR2F1 axis enhances cisplatin resistance in NSCLC, with NR2F1 identified as a crucial downstream mediator of NAS1 in this process (Figure 4). However, whether dormancy plays a role in cisplatin resistance in NSCLC through the NAS1-NR2F1 axis requires further experimental validation. Intriguingly, while NAS1 regulates cisplatin resistance through translational control of NR2F1, cisplatin-resistant cell lines exhibited reductions in both NR2F1 protein and NR2F1 mRNA levels (Figure 3B). This observation implies that NR2F1 downregulation in resistant cells may involve supplementary upstream regulatory mechanisms independent of NAS1, underscoring the intricate molecular circuitry underlying cisplatin resistance.
Transforming growth factor-β (TGF-β) is a pleiotropic cytokine implicated in multiple cellular processes—including cell development, proliferation, epithelial–mesenchymal transition (EMT), and immune regulation—via SMAD-dependent and non-SMAD signaling cascades (encompassing PI3K/AKT, MAPK, and NF-κB pathways) [24]. Intriguingly, TGF-β signaling exhibits dichotomous functions in cancer: it serves as a robust tumor suppressor during early tumorigenesis by inducing apoptosis or cell cycle arrest, yet paradoxically facilitates advanced tumor transformation, progression, and metastasis through multidimensional mechanisms [24]. Although TGFβ1 demonstrates context-dependent roles in oncogenesis, accumulating evidence indicates that elevated TGFβ1 expression fosters chemoresistance, with distinct molecular mechanisms identified across various malignancies [16,18,25,26], notably including NF-κB pathway activation-mediated drug resistance [18]. Consistently, our findings reveal that diminished NAS1-NR2F1 axis derepresses TGFB1 transcription, resulting in its upregulation and subsequent NF-κB pathway activation (evidenced by markedly increased phosphorylated p65 levels) (Figure 5 and Figure 6). These observations were corroborated in cisplatin-resistant cell lines as well as in NAS1- or NR2F1-deficient cellular models, substantiating the relationship between TGFβ1 overexpression and enhanced chemotherapeutic resistance.
In summary, our findings underscore the pivotal role of the NAS1-NR2F1 axis in modulating cisplatin resistance in NSCLC. Downregulation of this axis derepresses TGFB1 transcription, leading to NF-κB pathway activation and consequent promotion of cisplatin resistance. NF-κB activation has been widely implicated in cisplatin resistance, in part through promoting pro-survival and anti-apoptotic signaling. While the precise transcriptional regulatory mechanisms through which NR2F1 governs TGFB1 expression warrant further elucidation, therapeutic interventions aimed at restoring NAS1 expression (such as nucleotide analog administration), suppressing TGFB1 activity, or antagonizing downstream NF-κB signaling—particularly in combination with cisplatin—may offer a promising strategy for overcoming drug resistance in this malignancy. Although this study has validated the proposed mechanism in cell-based assays, the key findings have not yet been confirmed in animal models. Further in vivo and clinical investigations will be necessary to substantiate the translational relevance of this regulatory axis.

5. Conclusions
In this study, we report a comprehensive signaling cascade underlying cisplatin resistance in NSCLC. We propose a model in which downregulation of NAS1 impairs the translation of NR2F1. The subsequent loss of NR2F1 relieves its transcriptional repression of TGFB1, leading to increased TGFB1 expression. Upregulated TGFB1 subsequently activates the downstream NF-κB pathway, ultimately driving the acquisition and maintenance of the cisplatin-resistant phenotype.