The E3 ubiquitin ligase RNF180 modulates the EGFR/PI3K/AKT pathway to reduce cisplatin resistance in non-small cell lung cancer.

Introduction
Globally, lung cancer continues to be the primary cause of mortality associated with cancer, especially in people aged 50 years and older1. Non-small cell lung cancer (NSCLC) accounts for approximately 80–85% of lung cancer cases. Yet, its five-year survival rate is grim, falling below 20%, primarily because of frequent diagnoses at advanced stages2. This unfavorable outcome is directly related to inherent drug resistance3. In a clinical context, only a limited number of NSCLC patients are diagnosed at early stages (I/II), which can be addressed through surgical intervention. Conversely, more than 60% of patients are identified at locally advanced or metastatic stages (III/IV), indicating the need for standard chemoradiotherapy. Although cisplatin-based combination chemotherapy is the established first-line treatment for advanced NSCLC, the effectiveness of this approach is significantly compromised by chemoresistance4. Cisplatin (CDDP) mainly induces cytotoxicity by forming DNA adducts, which activate the DNA damage response and lead to apoptosis. Nevertheless, severe drug resistance critically undermines clinical results5. The mechanisms underlying resistance encompass a range of processes, including diminished drug uptake, increased efflux, altered drug metabolism, target mutations, enhanced DNA repair capacity, decreased mismatch repair efficiency, and evasion of apoptosis6. Therefore, a thorough exploration of the mechanisms driving cisplatin resistance is critical.
Ring finger protein 180 (RNF180), a significant member of the E3 ubiquitin ligase family, incorporates Ring finger and coiled-coil domains within its structural composition7. As an essential component of the ubiquitin–proteasome system (UPS), RNF180 selectively identifies target proteins to facilitate their ubiquitination and subsequent degradation8. This mechanism allows RNF180 to influence vital biological processes, such as apoptosis, gene transcription, and DNA repair9. In the field of oncology, RNF180 has been recognized as a tumor suppressor in hepatocellular carcinoma10, gastric cancer11, and colorectal cancer12. Notably, in triple-negative breast cancer, RNF180 reduces cell sensitivity to gefitinib by downregulating RAD51 expression13. However, the molecular mechanisms by which RNF180 regulates cisplatin resistance in NSCLC have not been identified.
The epidermal growth factor receptor (EGFR), a glycoprotein located in the cell membrane, possesses an intracellular tyrosine kinase domain14. This tyrosine kinase is responsible for promoting cell proliferation, inhibiting apoptosis, and stimulating neovascularization, which collectively drive tumorigenesis, invasion, and metastasis15. Approximately 60% of NSCLC patients show EGFR upregulation, confirming its status as a critical marker for disease progression14,15. The PI3K/AKT pathway, a downstream mediator of EGFR, is involved in numerous malignancies16. It plays a crucial role in regulating oncogenic processes, including cell growth, apoptosis inhibition, extracellular signaling, migration, and resistance to treatment17. This pathway is activated in 90% of NSCLC cases and represents a significant therapeutic target for tumor development18. Overall, the EGFR/PI3K/AKT signaling pathway plays a crucial role in the proliferation and progression of cancer, and EGFR has been clinically confirmed as a therapeutic target in NSCLC19–22.
This study first elucidates the role of RNF180 in cisplatin resistance in NSCLC. Through bioinformatics analysis, PLK2 was identified as an RNF180 substrate implicated in platinum resistance. We further explored the molecular mechanisms underlying the synergistic regulation of cisplatin resistance by RNF180.

Materials and methods

Cell cultures and reagents
The NSCLC cell lines A549 and H1975, the cisplatin-resistant lung adenocarcinoma cell line A549/DDP, and the normal lung epithelial cell line BEAS-2B were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan). A549 and H1975 cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution (P/S). A549/DDP cells were maintained in Ham’s F-12 K medium (Gibco, USA) supplemented with 10% FBS,1 µg/mL cisplatin, and 1% P/S. BEAS-2B cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% FBS and 1% P/S. All the cell lines were incubated at 37 °C in a humidified atmosphere with 5% CO₂. Jiangsu Hansoh Pharmaceutical Group Co., Ltd, provided Cisplatin.

Gene overexpression and knockdown
The lentiviruses for RNF180 overexpression LV5-RNF180(oe-RNF180) and its negative control LV5NC(NC5), as well as the three PLK2 knockdown lentiviruses (LV3-PLK2-2115 [siP2], LV3-PLK2-412 [siP4], LV3-PLK2-618 [siP6]) and their negative control LV3NC (NC3), were synthesized by GenePharma (Suzhou, China). When the A549/DDP cells reached 80% confluence, they were transfected with the corresponding lentiviruses. Specifically, cells transfected with LV5-RNF180 and NC5 were incubated with the lentivirus and an infection enhancer for 72 h, while cells transfected with LV3NC, LV3-PLK2-2115, LV3-PLK2-412, and LV3-PLK2-618 were incubated with the lentivirus and an infection enhancer for 48 h.

Real-time quantitative reverse transcription PCR (qRT-PCR)
Total RNA was extracted from cells and nude mouse tissues via TRIzol reagent (Takara, Japan). The extracted RNA was reverse-transcribed into cDNA with the TaKaRa PrimeScript™ RT Master Mix Kit (Takara, Cat. No. RR036A) under the following reaction conditions: 37°C for 15 minutes, 85°C for 5 seconds, and maintained at 4°C. The synthesized cDNA was stored at -80°C for subsequent use. For qRT-PCR validation, analysis was performed via the TB Green Premix Ex Taq™ kit (Takara, Cat. No. RR420A). The relative expression levels were determined via the 2−△△Ct method. The primer sequences used in this study were as follows: RNF180 Forward: 5’-GGCAGGCAGACTAATGAGACCATC-3’, RNF180 Reverse: 5’-AGCCACCACCTGTCAGCAGAG-3’, GAPDH Forward: 5’-AGGTCGGTGTGAACGGATTTG-3’, GAPDH Reverse: 5’-TGTAGACCATGTAGTTGAGGTCA-3’, PLK2 Forward: 5’-ACCTCATCAAAGGGAAAAGATTGAC-3’, PLK2 Reverse: 5’-CTTCTGGCTCTGTCAACACCT-3’.

Western blotting
BEAS-2B, A549, A549/DDP, and H1975 cells were scraped into the medium and centrifuged at 1500 relative centrifugal force (rcf) at 4 °C for 5 min, after which the cell pellets were collected. Proteins were extracted following cell lysis via RIPA buffer supplemented with PMSF. Protein concentrations were determined via a BCA assay (Beyotime Biotech Co., Ltd.). SDS‒PAGE was used to separate equal amounts of protein, which were then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% skim milk. The primary antibodies used were anti-RNF180 (Proteintech, 30941-1-AP), anti-E-cadherin (Abcam, Ab231303), anti-Vimentin (Abcam, Ab8069), anti-Ki67 (Abcam, Ab16667), anti-Caspase-9 (Proteintech, Ab202068), anti-P-gp (Proteintech, 22336-1-AP), anti-LRP (Proteintech, 65594-1-MR), anti-EGFR (Proteintech,30847-1-AP), anti-p-EGFR (Proteintech, 30277-1-AP), anti-p-PI3K (Proteintech, 67071-1-Ig), anti-PI3K (Abcam, ab302958), anti-p-AKT (Proteintech, 80455-1-RR), anti-AKT (Proteintech, 10176-2-AP), and anti-GAPDH (Proteintech, 60004-1-Ig). The secondary antibodies used were goat anti-rabbit IgG (Proteintech, SA00001-2) and goat anti-mouse IgG (Proteintech, SA00001-1).

Colony formation assay
When the cells in each group reached 90% confluence, they were digested with trypsin, centrifuged, and collected. The supernatant was discarded, and the cells were resuspended in complete culture medium for counting. For the colony formation assay, lentivirus-transfected cells were seeded at a density of 800 cells/well in 6-well plates and incubated at 37 °C in a 5% CO₂ incubator. After 8 days, the viable colonies were washed with phosphate-buffered saline (PBS) and stained with crystal violet solution (Beyotime).

Flow cytometry assay
The cells were seeded in 6-well plates at 2 × 10⁵ cells/mL and incubated for 24 h until they reached 70%–90% confluence. After 3 washes with precooled PBS, the cells were resuspended at a density of 1 × 10⁶ cells/mL. 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) (Vazyme, A211-01) were added, mixed well, and incubated in the dark for 15 min. Apoptosis was detected via flow cytometry. The cells were seeded in 12-well plates, and upon reaching 70–80% confluence, different concentrations of 0, 1, 2, and 4 µg/mL cisplatin were added, followed by 48 h of incubation. After 3 washes with precooled PBS, the cells were resuspended at a density of 1 × 10⁶ cells/mL. 5 µL of Annexin V-APC and 5 µL of PI (Annexin V-APC/PI Apoptosis Detection Kit -A214) were added, mixed well, and incubated in the dark for 10 min. Apoptosis was analyzed via flow cytometry.

Migration and invasion assays
Cell migration ability was evaluated via transwell chambers with an 8 μm pore size (Corning, USA). 16 h after transfection, A549/DDP cells were digested with trypsin and resuspended in an appropriate amount of serum-free F12K medium (Gibco). Subsequently, 2 × 10⁵ cells in F12K basal medium were seeded into the upper chamber, while F12K medium containing 10% fetal bovine serum was added to the lower chamber. After 48 h, non-migrated cells on the surface of the transwell chamber were removed with a cotton swab. The transwell membranes were fixed in 4% paraformaldehyde for 20 min, air-dried, and then stained with 0.1% crystal violet solution (Beyotime) for 30 min. The number of cells that had migrated through the membrane to the lower surface was observed under an optical microscope and counted via ImageJ software. The procedure for the invasion assay was the same as that for the cell migration assay, except that a diluted layer of Matrigel (Vazyme, GL101-01) was precoated on the membrane filter before the experiment.

Xenograft tumor models
Ten 4-to-5-week-old, SPF-grade male BALB/c nude mice (purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd., license number: SCXK (Zhe) 2024–0004) with a body weight of 18–24 g were used in this study. The mice were randomly divided into two groups (5 mice per group) to eliminate the influence of body weight differences. All mice were housed in a clean environment with constant temperature and humidity, maintained at 25 ± 1 °C and 70%, respectively, under a 12-hour light/dark cycle. The mice had free access to standard feed and sterilized water. On the 7th day after the end of the quarantine period, the mice were subjected to subcutaneous injection for inoculation. After pre-culturing the cells, they were inoculated into the axillary subcutaneous tissue of each mouse at a density of 5 × 10⁶ cells/mL, with an inoculation volume of 200 µL per mouse. The state of the animals was observed daily, and the tumor volume was measured once every 3 days, for a total of 9 measurements. The tumor diameters of the mice in each group were measured with vernier calipers, the tumor volumes were calculated, and changes in body weight were recorded. The tumor volume (V) was calculated using the following formula: V = 1/2×a×b² (where V represents the tumor volume in mm³; a is the long diameter of the tumor in mm; b is the short diameter of the tumor in mm), and the maximum tumor volume did not exceed 2000 mm³. Isoflurane inhalation anesthesia (2.5%) was administered before tumor tissue excision, and the mice were subsequently sacrificed by cervical dislocation. A portion of the tumor tissues was processed into tissue lysates for Western blot analysis, while another portion was sectioned into tissue slices. Immunohistochemical (IHC) staining was executed in strict adherence to the manufacturer’s protocol. All primary antibodies employed are specified in the Western blotting section. All animal experimental protocols obtained approval from the Ethics Committee of Eastern Theater Command General Hospital (Approval No. DZGZRDW240010) and were conducted in strict conformity with the committee’s relevant guidelines and regulations. The implementation of this study adheres to the ARRIVE guidelines (Animal Research: Reporting of In vivo Experiments).

Hematoxylin‒eosin (HE) staining
Tissues were fixed with a paraformaldehyde fixative (Shanghai Macklin, P804536). Hematoxylin solution (Shanghai Macklin, H810910) and a 1% alcohol-soluble eosin solution (Shanghai Macklin, E809023) were used for staining. After graded ethanol dehydration (Shanghai Macklin, E809056), xylene clearing (Shanghai Macklin, X820585), and mounting with neutral balsamum (Solarbio, G8590) were performed on the tissue sections. The stained sections were observed under a microscope.

Statistical analysis
Statistical analysis was performed via GraphPad Prism v.10.1.2 (Windows version). The experimental data are presented as the means ± standard deviations (SDs) of at least 3 biological replicates. One-way analysis of variance (one-way ANOVA) or unpaired Student’s t-test was used to determine statistical significance, with a p-value < 0.05 considered statistically significant.

Results

RNF180 is downregulated in A549/DDP cells, and its low expression is associated with poor prognosis in NSCLC patients
To clarify the significance of RNF180 in the pathogenesis of NSCLC, we investigated its expression via the TIMER2.0 database (http://cistrome.org/TIMER/). Across various malignancies, including NSCLC, RNF180 was found to be significantly downregulated (p < 0.001) (Fig. 1A). We examined RNF180 expression in lung adenocarcinoma (LUAD) tumors and normal samples from The Cancer Genome Atlas (TCGA) (https://www.cancer.gov/ccg/research/genome-sequencing/tcga). The analysis revealed that RNF180 expression was significantly lower in LUAD samples compared to normal samples (p < 0.001) (Fig. 1B–C). In contrast to previous investigations, NSCLC cell lines (A549, A549/DDP, and H1975) presented significantly higher RNF180 mRNA levels compared with normal BEAS-2B cells (p < 0.0001) (Fig. 1D). Conversely, Western blotting demonstrated substantially reduced RNF180 protein expression in NSCLC lines, with the most pronounced decrease in A549/DDP cells (Fig. 1E). Subsequent qRT-PCR and Western blotting validation confirmed significantly lower RNF180 mRNA (p < 0.0001) and protein (p < 0.01) levels in A549/DDP cells than in A549 cells (Fig. 1F–G). By querying the TCGA database, we found that low RNF180 expression correlated with poor overall survival (OS) (n = 530, p = 0.010, HR = 0.69) in NSCLC patients (Fig. 1H).

RNF180 overexpression suppresses proliferation, migration, invasion, EMT, cisplatin resistance, and metastasis both in vitro and in vivo
To investigate the regulatory effect of RNF180 on cisplatin sensitivity in A549/DDP cells, RNF180-overexpressing lentivirus (oe-RNF180) and a negative control lentivirus (NC5) were constructed and transduced into A549/DDP cells. The successful overexpression of RNF180 was confirmed by detecting significantly increased protein and mRNA levels through Western blotting (p < 0.001) and qRT-PCR (p < 0.0001) (Fig. 2A-C). We subsequently evaluated the biological functions of RNF180 via both in vitro assays and in vivo tumor xenograft models. Colony formation assays revealed that RNF180 overexpression markedly inhibited colony formation ability (p < 0.05) (Fig. 2D-E). Transwell assays showed that RNF180 overexpression suppressed the migration and invasion of A549/DDP cells (p < 0.0001) (Fig. 2F-H). Furthermore, compared with the NC5 group, RNF180 overexpression significantly promoted apoptosis in A549/DDP cells. Specifically, the apoptotic rate was significantly higher in oe-RNF180 cells (9.86% ± 5.57%) than in NC5 cells (7.76% ± 1.59%)( p < 0.001) (Fig. 2I-J). Western blotting analyses of EMT markers revealed that RNF180 overexpression significantly upregulated E-cadherin protein (p < 0.001) but downregulated Vimentin protein (p < 0.01) (Fig. 2K-M). The downregulation of the drug resistance proteins P-glycoprotein (P-gp) (p < 0.0001) and LRP (p < 0.01) was observed in RNF180-overexpressing A549/DDP cells (Fig. 2N-P). Furthermore, we explored the role of RNF180 in vivo. Stably manipulated A549/DDP cell lines were obtained via the use of RNF180-overexpressing or NC5 lentivirus. In the tumor xenograft experiments (n = 5 per group), RNF180 overexpression in A549/DDP cells significantly slowed tumor growth, with both tumor volume (p < 0.001) and weight (p < 0.001) markedly reduced (Fig. 2S-V). Moreover, compared with those in the NC5 group, the protein (p < 0.001) expression levels of E-cadherin were greater, and the protein (p < 0.01) expression levels of Vimentin were lower (Fig. 2Q-R). Furthermore, immunohistochemistry revealed that RNF180 could increase the number of caspase-9-positive cells to promote apoptosis and reduce the number of Ki67-positive cells to inhibit proliferation. (Fig. 2W).

RNF180 overexpression suppresses EGFR/PI3K/AKT signaling activity
To elucidate the mechanism of RNF180 in NSCLC, we carried out functional enrichment analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses (www.kegg.jp/kegg/kegg1.html) revealed that RNF180 was enriched in pathways related to post-transcriptional mRNA repression and trans-splicing (Fig. 3A). Moreover, gene set enrichment analysis (GSEA) highlighted the critical involvement of genes associated with RNF180 in signaling pathways, including protein kinase A (PKA)-mediated CREB phosphorylation, DAG-IP3, MYC, and epidermal growth factor receptor (EGFR) (Fig. 3B). In A549/DDP cells, RNF180 overexpression significantly inhibited the EGFR/PI3K/AKT signaling pathway, which specifically resulted in a marked reduction in the phosphorylation of proteins, including p-PI3K (p < 0.01), p-AKT(p < 0.01), and p-EGFR(p < 0.01) (Fig. 3C-G). To further validate this suppressive effect in vivo, similar analyses were performed on nude mouse models. RNF180 overexpression also inhibited phosphorylation-dependent activation of PI3K(p < 0.01), AKT(p < 0.01), and EGFR(p < 0.001), confirming that RNF180 suppresses the EGFR/PI3K/AKT pathway in vivo (Fig. 3H-L). Collectively, these results indicate that RNF180 attenuates cisplatin resistance in NSCLC by inhibiting the EGFR/PI3K/AKT signaling pathway.

PLK2 may serve as a potential substrate of the E3 Ubiquitin Ligase RNF180
To identify potential downstream substrates that mediate RNF180’s regulatory function in NSCLC, we initially predicted the potential interacting proteins of RNF180 using the STRING database (https://cn.string-db.org), and the results indicated its association with several candidate proteins, including PLK2 and ZIC2 (Fig. 4A). We further screened the co-expression gene network of RNF180 via the BioGRID database, which validated the interaction between PLK2/ZIC2 and RNF180 (Fig. 4B). Heatmap analysis demonstrated that RNF180 was significantly associated with genes representative of AMELX(p < 0.001), RBPQX(p < 0.001), MAB21L2 (p < 0.01), and PLK2 (p < 0.01) (Fig. 4C). We further investigated the correlation between RNF180 and genes of the PLK family. The results showed that RNF180 expression was significantly correlated with PLK2 expression (p < 0.01) (Fig. 4D). Meanwhile, data from GEPIA (http://gepia.cancer-pku.cn/) confirmed a positive correlation between RNF180 and PLK2 in NSCLC, as demonstrated by Pearson correlation analysis (R = 0.32, p = 0) and Spearman correlation analysis (R = 0.43, p = 8.4e-38) (Fig. 4E-F). Consistent with the bioinformatics predictions, RNF180 overexpression in A549/DDP cells significantly elevated PLK2 protein expression (p < 0.001). Conversely, PLK2 knockdown reduced RNF180 protein expression (p < 0.01) (Fig. 4G‒J). As RNF180 functions as an E3 ubiquitin ligase—a class of enzymes that typically mediate the ubiquitination of substrate proteins—analyses using the ubiquitination-focused databases UbiBrowser 2.0 and Ubipriot (http://ubibrowser.bio-it.cn/ubibrowser/home/index), combined with prior studies23, collectively suggest that RNF180 modulates PLK2 expression via ubiquitination (Fig. 4K‒L). Notably, considering the established role of PLK2 in cancer platinum resistance, these findings further support PLK2 as a functional interacting protein and putative substrate of RNF180.

Silencing of PLK2 promotes resistance of A549/DDP cells to cisplatin
Analysis of the TIMER 2.0 database confirmed that PLK2 expression was significantly reduced in LUAD and LUSC tumors relative to adjacent normal lung tissues(Fig. 5A). The protein and mRNA levels of PLK2 were significantly higher in A549/DDP cells than in A549 cells (p < 0.05) (Fig. 5B-C).To explore the biological function of PLK2 in cisplatin-resistant lung cancer cells, PLK2 was knocked down in A549/DDP cells via lentiviral transduction, with NC3 serving as the negative control. The results from qRT-PCR revealed that si-P2, si-P4, and si-P6 substantially decreased PLK2 expression, with si-P4 showing the most effective silencing (p < 0.0001) (Fig. 5D). Western blot analysis confirmed the knockdown efficacy of si-P4 (p < 0.001), which was consequently selected for subsequent experiments (Fig. 5E). The role of PLK2 in A549/DDP cells was assessed via colony formation assays (Fig. 5F-G), flow cytometry (Fig. 5H-I), and Transwell migration/invasion assays (Fig. 5L-N). Compared with NC3, PLK2 knockdown significantly increased cell proliferation (p < 0.05) and reduced apoptosis (p < 0.001). Transwell assays indicated that PLK2 knockdown increased cell migration considerably (p < 0.0001) and invasion (p < 0.001)(Fig. 5J-L). To specifically examine the role of PLK2 in acquired cisplatin resistance in A549/DDP cells, CCK-8, Transwell, and flow cytometry analyses were performed. After treatment with cisplatin at concentrations of 0, 1, and 2 µg/mL, cell viability in the PLK2-knockdown group was significantly higher than in the NC3 control. However, no statistically significant difference was detected at the 4 µg/mL cisplatin (Fig. 5M). Transwell assays further demonstrated that increasing cisplatin concentrations to 1 and 2 µg/mL significantly enhanced the number of migrating and invading cells in the PLK2 knockdown group, with no significant difference observed at 4 µg/mL (Fig. 5N‒O). Flow cytometry analysis revealed that the apoptotic rate of the PLK2 knockdown group (11.73%) was slightly lower than that of the NC3 group (12.07%) at 0 µg/mL cisplatin. After treatment with 1, 2, and 4 µg/mL cisplatin, the apoptotic rates of the PLK2 knockdown group were significantly reduced to 22.99%, 33.30%, and 42.30%, whereas those of the NC3 group were 37.02%, 43.00%, and 46.10%, respectively (Fig. 5P).

Discussion
RNF180 is expressed at low levels in several types of malignancies, including colorectal cancer12, breast cancer13, gastric cancer24, and non-small cell lung cancer(NSCLC)25. However, studies investigating RNF180 expression in NSCLC cell lines have reported inconsistent findings. In the present study, we utilized BEAS-2B cells as a control group and found that RNF180 mRNA levels were significantly elevated in A549, A549/DDP, and H1975 NSCLC cell lines, whereas RNF180 protein levels remained relatively low. In contrast, other studies that used human bronchial epithelial (HBE) cells as controls reported notable downregulation of both RNF180 mRNA and protein in five NSCLC cell lines, including A549 and H358, with particularly marked decreases observed in H1975 and H358 cells26. Similarly, research using MRC-5 controls confirmed lower RNF180 mRNA and protein expression in A549 and HCC827 cells than in nontumor cells25. These discrepancies may be due to two main factors. The first factor is that variations in control cell types may influence the results of mRNA detection. The second factor is that this discrepancy could be linked to the post-translational regulation of RNF180, an observation supported by various molecular mechanisms. For instance, E3 ligases show spatiotemporal variability between mRNA and protein levels during differentiation—such as the “translational delay” of ZEB2 in mouse gastruloid differentiation, where mRNA expression precedes protein accumulation27—a regulatory pattern that may also exist in tumor-related gene expression. Moreover, integrated transcriptomic and proteomic analyses in tumor cells or stress-responsive systems frequently reveal a lack of agreement between overall mRNA and protein expression levels28, which may contribute to the observed inconsistencies in RNF180 expression. In the present study, we demonstrated that RNF180 overexpression inhibits proliferation, migration, and invasion, while promoting apoptosis, in cisplatin-resistant NSCLC cells, thereby attenuating their cisplatin resistance; in vivo experiments further confirmed that RNF180 exerts a suppressive effect on tumor growth. Additionally, the downregulation of RNF180 is strongly associated with poor prognosis, advanced T stage, and increased N stage in NSCLC patients, highlighting its potential as a prognostic biomarker for disease progression24. This finding underscores the clinical significance of RNF180 and should be further verified with larger numbers of NSCLC cases.
Importantly, it is essential to further explore the underlying molecular mechanisms through which RNF180 exerts its function. As a key process regulating tumor invasion, metastasis, and chemoresistance, EMT is a key contributor to drug resistance in NSCLC29. Multidrug resistance (MDR) frequently coincides with EMT in tumors. Studies indicate that the expression levels of the drug resistance-related protein P-glycoprotein (P-gp) negatively correlate with chemosensitivity30–32, and multidrug-resistant tumor cells exhibiting high P-gp expression demonstrate enhanced invasion and metastatic capacity33. In the present study, we confirmed that RNF180 overexpression could inhibit EMT-mediated cell migration and invasion, downregulate the expression of the drug resistance-related proteins P-gp and lung resistance-related protein (LRP), and thereby increase cellular sensitivity to cisplatin. Furthermore, our data showed that KEGG34,35 and GSEA enrichment analysis revealed that RNF180 is enriched in the EGFR pathway, which prompted us to further explore the association between them. EGFR, a transmembrane glycoprotein critical in tumorigenesis, activates intracellular cascades, including the PI3K/AKT cascade, driving proliferation, invasion, and metabolic reprogramming, thereby establishing it as a key therapeutic target36–38. As a core effector of EGFR, PI3K/AKT regulates cell cycle progression, angiogenesis, epithelial-mesenchymal transition (EMT), and chemoresistance, with frequent aberrant activation observed in malignancies such as breast, ovarian, and lung cancers39,40. Previous studies on pathway regulation have shown that miR-1299 inhibits the migration and EMT of NSCLC cells by targeting EGFR41, while miR-133b overexpression blocks the PI3K/AKT and JAK2/STAT3 pathways, suppressing cisplatin-induced proliferation42. In this study, both in vitro and in vivo experiments confirmed that RNF180 overexpression significantly downregulates the protein expression levels of p-PI3K, p-AKT, and p-EGFR. These results confirm that RNF180 attenuates cisplatin resistance in NSCLC by regulating the EGFR/PI3K/AKT signaling pathway.
Ubiquitination is a crucial post-translational modification that conjugates ubiquitin molecules to lysine residues of target proteins via E3 ubiquitin ligases, thereby regulating the stability, subcellular localization, and biological functions of target proteins43. As an E3 ubiquitin ligase, RNF180 is functionally capable of mediating ubiquitination of substrate proteins. In the present study, analysis using the ubiquitination database revealed that RNF180 could mediate the ubiquitination of PLK2, while expression correlation analysis based on the GEPIA database demonstrated a significant positive correlation between RNF180 and PLK2 expression. Our cellular experimental results further verified that PLK2 expression was markedly upregulated in A549/DDP cells with RNF180 overexpression, whereas RNF180 expression was decreased following PLK2 knockdown. This finding is clearly inconsistent with the classical protein degradation paradigm mediated by ubiquitination. The linkage type of ubiquitin chains is the core determinant of their biological effects: K48-linked ubiquitin chains mainly mediate the proteasomal degradation of target proteins through recognition by ubiquitin-binding domains, while K63-linked ubiquitin chains are involved in the regulation of non-degradative biological processes, including signal transduction, protein trafficking, immune response, and DNA damage repair44. Previous studies have confirmed that listerin-mediated K63-linked ubiquitination of ABCA1 prevents lysosomal degradation by reducing its binding to HRS (an ESCRT subunit), thereby stabilizing ABCA1 and maintaining its membrane localization45. Existing studies have also confirmed that RNF181 can directly bind to YAP protein, inhibit its K48-linked polyubiquitination modification and subsequent proteasomal degradation, stabilize YAP protein levels, and further enhance the expression of its downstream target genes (such as CTGF and CYR61). Ultimately, this promotes the proliferation, migration, and invasion of triple-negative breast cancer (TNBC) cells and accelerates tumor progression46. Based on these findings, we hypothesize that RNF180-mediated ubiquitination of PLK2 may regulate PLK2 protein stability and biological activity by either activating the formation of non-degradative ubiquitin chains (such as K63-linked type) or inhibiting the formation of K48-linked degradative ubiquitin chains. This potential regulatory mechanism requires further experimental verification in subsequent studies.
PLK2, a member of the polo-like serine/threonine kinase family47, regulates cell cycle progression and DNA damage responses48. In this study, PLK2 expression was significantly upregulated in cisplatin-resistant A549/DDP cells compared with parental A549 cells. Functional analyses demonstrated that PLK2 knockdown in A549/DDP cells significantly enhanced clonogenic capacity, migration, and invasion while suppressing apoptosis, thereby promoting cellular chemoresistance to cisplatin. These findings align with the well-documented dual roles of PLK2 in tumors: in glioblastoma (GBM), for instance, PLK2 expression is notably lower in tumor tissues than in normal brain tissues, and it acts as a tumor suppressor during the initiation and progression of primary GBM23. Conversely, the loss of PLK2 has been shown to activate the Notch signaling pathway by negatively regulating HES1 transcription and promoting the ubiquitin-dependent degradation of Notch1, which in turn enhances tumor cell invasiveness and induces acquired resistance to temozolomide (TMZ)23. Additionally, PLK2 expression is regulated by microRNAs (e.g., miR-126) and epigenetic mechanisms (such as CpG island methylation in the promoter region), and these regulatory patterns are closely associated with chemosensitivity and clinical prognosis across various tumor types23. Additionally, under cisplatin intervention, specifically when the cisplatin concentration was increased to 1 µg/mL and 2 µg/mL, the PLK2 knockdown groups exhibited higher cell viability, enhanced migratory and invasive capacities, and significantly reduced cisplatin-induced apoptosis. These effects collectively reverse the inhibitory effects of cisplatin, thereby enhancing the resistance of A549/DDP cells to cisplatin. These results further confirm the critical role of PLK2 downregulation in promoting cisplatin resistance. Thus, targeting PLK2 represents a promising therapeutic strategy, particularly for patients with chemoresistant tumors.
However, this study has several limitations. First, although the protein–protein interaction between RNF180 and PLK2 has been predicted by multiple bioinformatics databases, it remains unvalidated by co-immunoprecipitation (CO-IP) experiments. Second, while a positive correlation between RNF180 and PLK2 has been confirmed via in vitro and in vivo experiments as well as bioinformatics analyses, the specific mechanism underlying RNF180-mediated PLK2 ubiquitination remains unelucidated, necessitating further experimental validation.

Conclusion
Collectively, our data demonstrate that RNF180 inhibits proliferation, migration, invasion, and EMT while promoting apoptosis in A549/DDP cells. Mechanistically, RNF180 attenuates cellular cisplatin resistance by suppressing the EGFR/PI3K/AKT signaling pathway. Furthermore, we identified PLK2 as a substrate associated with RNF180-mediated ubiquitination, which plays a critical role in mediating cisplatin resistance in NSCLC. These findings provide novel mechanistic insights for reducing chemoresistance in NSCLC.

Supplementary Information
Below is the link to the electronic supplementary material.