LINC00467 mediates PARP inhibitor resistance in high-grade serous ovarian cancer by regulating miR-485-5p/RAD50 axis

Abstract

Purpose

High-grade serous ovarian cancer (HGSOC) is extremely lethal owing to its high risk of recurrence and resistance to common chemotherapy. PARP inhibitor (PARPi) is currently the most promising target drug among patients with HGSOC, but its resistance mechanism remains unclear. In this study, we aimed to investigate the mechanism of PARPi resistance and explore new therapeutic targets.

Methods

We established olaparib-acquired resistant cell lines denoted SKOV3-OR and performed lncRNA sequencing. Biological behavior study illustrated the effect of LINC00467 on HGSOC cells in vitro and vivo. Bioinformatics and the dual-luciferase reporter system confirmed the downstream target genes. Patient-derived xenograft (PDX) models were established to validate the effect of LINC00467 on response to olaparib.

Results

LncRNA sequencing revealed that LINC00467 was upregulated in PARPi resistant HGSOC cell and predicted poor prognosis. Biological behavior study comfirmed LINC00467 promoted cell proliferation, metastasis and PARPi resistance in HGSOC. Furthermore, the dual-luciferase reporter system confirmed its function as a competitive endogenous RNA by directly sponging miR-485-5p, which further regulated its target, RAD50. Additionally, LINC00467 ASO played a synergistic role with olaparib in HGSOC PDX models.

Conclusion

Our results showed that LINC00467 could confer PARPi resistance in HGSOC by regulating the miR-485-5p/RAD50 axis and provide a novel therapeutic target in PARPi synergistic treatment for HGSOC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13048-026-01971-9.

Introduction

High-grade serous ovarian cancer (HGSOC) is a common malignant tumor in women with a high lethal rate (90%) and its 5-year survival rate is less than 30% [1] because most patients are diagnosed at an advanced stage with frequent metastasis [2]. Moreover, almost 70% of patients relapse, although they have accepted standard cytoreductive surgery and chemotherapy because of chemotherapy resistance [3, 4]. Lu J et al. suggests that the prognosis of ovarian cancer patients may be related to Oxidative Phosphorylation (OXPHOS) [5]. Their study revealed that OXPHOS-related genes were considerably abundant in a series of immune processes, suggesting a better response to immune checkpoint inhibitors. However, the mechanisms underlying the response and resistance to immune checkpoint inhibitors in ovarian cancer patients still need further investigation. Thus, exploring more specific and sensitive therapeutic targets, is a continuous direction and hotspot in ovarian cancer research.

Poly ADP-ribose polymerase inhibitor (PARPi) is the first Food and Drug Administration (FDA)-approved target drug for patients with HGSOC based on its genomic characteristics because of its impressive results in clinical trials [6]. Homologous recombination repair (HRR) was defective in BRCA mutation (BRCAm) cells. Single-stranded DNA breaks cannot be repaired and accumulated continuously when treated with PARPi. The risk of double-stranded DNA breaks that were mainly repaired through HRR increased, resulting in cytotoxicity and synthetic lethality [7]. Although PARPi has been accepted as a maintenance or late-line treatment for HGSOC, 40–70% of patients with BRCAm show acquired resistance or primary insensitivity [8, 9]. Consequently, researchers’ footsteps include investigating the preclinical prediction of PARPi response and exploring potential drug resistance mechanism to benefit more patients with HGSOC.

Long non-coding RNAs (lncRNAs) are more than 200 nucleotides in length with or without protein-coding capacity and have been proven to be key regulators of target gene expression in various biological processes, such as gene transcription, RNA splicing, and RNA transport and translation [10, 11]. The abnormal expression of lncRNAs has been found to be involved in oncogenesis and development [12] by functioning as competing endogenous RNAs (ceRNAs) via sponging microRNAs (miRNAs) and regulating the expression of specific genes [13]. Xu J et al. [14] have proven that UCA1 functions as an oncogene in ovarian cancer. The researchers found that knockdown of UCA1 inhibited ovarian cancer cell viability, which a miR-99b-3p inhibitor could reverse in vitro. Their work contributed in the pathogenesis of OC that the lncRNA-related network mechanisms were involved in. This research also provide strong evidence that (CRISPR)-caspase 9 (Cas9), has the potential to treat ovarian cancer via gene editing strategies [15]. For instance, upregulation of the lncRNA SOCAR promotes proliferation, migration, and invasion in ovarian cancer cells [16]. The lncRNA NEAT1 has been shown to promote cell proliferation and migration by sponging miR-506 in HGSOC [17]. In this study, we investigated the lncRNAs that mediate the response to PARPi in HGSOC and their relevant downstream signal transduction mechanisms to explore latent synergistic therapeutic targets.

In the present study, we established a PARPi-resistant cell line and performed lncRNA sequencing to identify a novel lncRNA associated with PARPi responses and HGSOC patient’ prognosis. Further molecular biology studies revealed the potential mechanisms by which LINC00467 confers PARPi resistance in HGSOC by regulating the miR-485-5p/RAD50 axis. Most importantly, we established patient-derived xenograft (PDX) models and validated this hypothesis in patient-derived tumor tissues. These findings provide new insights into the molecular functions of LINC00467 and indicate a novel target for a PARPi synergistic therapeutic strategy in HGSOC.

Materials and methods

Tumor tissues

Sixty HGSOC tissues with relevant clinical data were collected from Qilu Hospital of Shandong University. All enrolled patients were followed for at least 5 years. The tumor stage was determined according to the FIGO staging system. Tissues RNA was extracted from the formalin-fixed, paraffin-embedded (FFPE) samples using the AllPrep DNA/RNA FFPE Kit (QIAGEN) according to the manufacturer’s recommendations. This study was approved by the ethic committee of Qilu Hospital of Shandong University.

PARPi-resistant inducement and LncRNA sequencing analysis

SKOV3 cells were continuously incubated with olaparib at half-IC50 concentration for at least 3 months to induce the PARPi-resistant cell line SKOV3-OR, whose IC50 was three times more than that of the original SKOV3. lncRNA sequencing was conducted using the Illumina PE150 system (Novogene, Beijing, China). The fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) value was obtained to represent the expression level of certain genes. The differential analysis of lncRNA expression between SKOV3 and SKOV3-OR is illustrated in a heat map using GraphPad Prism 8 (GraphPad Software, USA). P < 0.05 and log2 fold change absolute value > 1 were set as the threshold for significantly differential expression default. Raw sequencing data were uploaded to the GEO repository (GSE208356).

Quantitative RT-PCR (qRT-PCR) analysis

Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed using a PrimeScriptTM RT Reagent Kit (TaKaRa Bio, China) following the manufacturer’s instructions. qRT-PCR was performed using the SYBR Premix Ex Taq™ Kit (Takara). U6 or β-actin was used as the internal control. Each analysis was performed in triplicates. Relative expression was determined using the 2-∆∆CT method. The primers were synthesized by GenePharma (Shanghai, China).

Cell culture and proliferation

A2780 and SKOV3 were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37℃. The cell proliferation assay was performed using 3-(4, 5)-dimethylthiazo (-z-y1)−3, 5-diphenytetrazoliumromide (MTT; Sigma-Aldrich) and a clonogenic assay. For cell viability detection, cells were seeded in 96-well plates and incubated at 37℃ for 0–5 days. The MTT solution was added, and the plate was incubated for 5 h. Later, the supernatants in the wells were discarded and 100µL DMSO (Sangon Biotech) was added to measure the absorbance at 490 nm using a microplate reader (Bio-Rad). For the clonogenic assay, single-cell solutions were plated onto 6-well plates and incubated at 37℃ for 12–14 days. Colonies were fixed with methanol and stained with 0.1% crystal violet. Colonies containing > 50 cells were counted.

Transwell assay

Cell migration and invasion were assessed using a Transwell system (Corning, NY, USA). For the invasion assay, matrigel (Corning) was added to the upper chamber. After incubation, the cells in the upper chamber were removed, and the cells on the lower surface were fixed with 20% methanol and stained with 0.1% crystal violet. The cells were observed and counted under a microscope.

Cell transfection

The vector stably overexpressing LINC00467 and the corresponding negative control were constructed by Genechem (Shanghai, China) and then transfected via a lentivirus-packaging system. Small interfering RNA (siRNA) targeting LINC00467 (sequence: 5’-TCAGACTCATGAAACCAAT-3’), miR-485-5p mimics, miR-485-5p inhibitor and controls were designed and synthesized by GenePharma (Shanghai, China).A2780 and SKOV3 cells were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA).

Western blotting

Cells were lysed suing radioimmunoprecipitation assay (RIPA) Lysis Buffer (Beyotime, China) and the protein concentration was detected using a BCA Assay Kit (Thermo Scientific). A total of 30 µg proteins were separated by SDS-PAGE (5% stacking gel and 12% separation gel) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) by Bio-Rad Trans-blot (15 V, 90 min). The membranes were blocked with 5% non-fat milk for 1 h and incubated in primary antibody solution overnight at 4 °C. The secondary antibodies were incubated for 1.5 h and then protein bands were detected using the Pro-lighting horseradish peroxidase (HRP) agent. β-actin was used as an internal control. The following primary antibodies were purchased from CST or Abcam: N-CAD (CST:13116), E-CAD (CST:3195), vimentin (CST:5741), β-catenin (CST:8480), CCND1 (CST:92G2), CDK4 (Abcam: Ab108357), CDK6 (Abcam: Ab124821), RAD50 (Abcam: Ab208019), and β-actin (CST:3700) and were 1:1000 diluted.

Drug resistance assay in vitro

Olaparib (AZD2281, Selleckchem, USA) was added into 6- or 96-well plate at various final concentrations in which different cells were seeded and incubated for 48–60 h. The final relative cell viability change was estimated using the MTT method, and the surviving colonies were calculated.

Subcellular fractionation

Nuclear and cytoplasmic RNA were fractionated using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific, USA). qRT-PCR was used to detect the relative RNA levels in the nucleus and cytoplasm. MALAT1 served as a positive nuclear control, whereas β-actin served as a cytoplasmic control. U6 was used as the nuclear control transcript.

Luciferase assay

The dual-luciferase pmirGLO vector (Promega, Madison, Wisconsin, USA) was used to generate luciferase reporter constructs. Wild-type (LINC00467 WT and RAD50 WT) and mutant (LINC00467 MT and RAD50 MT) vectors were constructed by GenePharma (Shanghai, China). HEK293T cells were co-transfected with miR-485-5p mimic and LINC00467 WT (or LINC00467 MT) vector using Lipofectamine 2000. Luciferase activity was examined using a luciferase reporter assay kit (TransGen Biotech). A similar process was also observed between the miR-485-5p mimics and RAD50 WT (or RAD50 MT) vectors. Each experiment was performed in triplicate.

Drug resistance assay in nude mice

Four weeks-old female athymic BALB/c nude mice were purchased from the Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China) and maintained in a pathogen-free facility. A total of 5 × 106 SKOV3 cells overexpressing LINC00467 or NC were resuspended in 200 µl PBS and injected into the bilateral armpit of each mouse (n = 10). Two weeks later, the mice were randomly divided into treated (n = 5) or untreated (n = 5) groups. The treated group received an intraperitoneal injection of olaparib (50 mg/kg) once a day for 2 weeks while the untreated group received DMSO dilution in the same mode. The tumors were harvested, photographed, and weighed to illustrate the growth of the different groups.The tumor tissues from each group were embedded in paraffin, sectioned, and subsequently subjected to immunohistochemical analysis using RAD50 antibodies to compare the expression levels of RAD50 across different groups. The obtained data were analyzed using the open-source bioimage analysis software QuPath (V.0.4.3). All animal experiments were performed with approval from Qilu Hospital of Shandong University Animal Care and Use Committee.

PDX model and therapy experiment

PDX model was used in this study. Fresh HGSOC tissues obtained from the Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, were cut into homogenates and injected subcutaneously into 4–6-weeks-old female NCG mice purchased from NBRI (China). When the tumors reached 10 × 10 × 10 mm (usually after approximately 2–3 months), they were harvested, homogenized, and injected intraperitoneally into new recipient mice. The antisense oligonucleotide (ASO) of LINC00467 was synthesized by RiboBio (Guangzhou, China). The olaparib/DDW or olaparib/ASO regimen was achieved in two PDX groups (each containing three mice). The number and weight of abdominal metastases in the different treated mice were recorded and compared. Paraffin-embedded sections of tumor tissues from different groups were prepared and immunofluorescence analysis of their foci was performed using RAD50 and γ-H2AX antibodies. All NCG mice were maintained in a SPF-level house. Qilu Hospital of Shandong University Animal Care and Use Committee approved the PDX model experiment for this study.

Statistical analyses

Each experiment was independently repeated in triplicate, (Even when conducting a complete experiment using three batches of independently revived cells with consistent passage numbers, each batch of cells is initiated from the thawing process and independently undergoes transfection, treatment, and detection). OS analysis was performed using Kaplan–Meier and log-rank tests. Values are shown as mean ± standard deviation (SD). Statistical analyses were performed using the GraphPad Prism 8 software (USA). Statistical significance was set at P < 0.05.

Results

LINC00467 is upregulated in PARPi resistant HGSOC cell and predicts poor prognosis

According to the lncRNA sequencing and bioinformatic analysis results, there were 11,283 and 10,674 upregulated and downregulated genes, respectively, between SKOV3 and SKOV3-OR (Table S1). The top 10 up-regulated and 10 down-regulated lncRNAs are shown in the heat-map (Fig. 1A). The volcano plot revealed the log2 fold change value alteration and –log adjusted P value of all differentially expressed genes (Fig. 1B).In the GEPIA gene expression profile, LINC00467 expression was elevated in most tumor tissues compared to that in the corresponding normal tissues (Fig. 1C). Consistently, we compared the expression of LINC00467 in OSE, FTE and ovarian cancer and revealed the significantly higher expression in ovarian cancer(Fig. 1D).The cohort of Qilu Hospital of Shandong University was divided into high and low LINC00467 groups based on the median relative expression value, between which the Kaplan-Meier Plotter demonstrated a significant overall survival difference. The overall survival rate of the high LINC00467 group was consistently lower than that of the low LINC00467 group (Fig. 1E). Furthermore, the HGSOC cohort from Qilu Hospital of Shandong University was divided into platinum-sensitive (n = 20) and platinum-resistant (n = 30) groups according to the platinum-free interval (6 months as threshold). Compared to the platinum-sensitive group, the platinum-resistant group expressed LINC00467 at a significantly higher level (P < 0.01) in the student’s t-test (Fig. 1F).

Fig. 1.

A. Heat map illustrated the 10 top increased lncRNAs and 10 top decreased lnRNAs between SKOV3-OR and SKOV3. B. The volcano plot revealed 11,283 up-regulated genes and 10,674 down-regulated genes with their log2 fold change value alteration and –log adjusted P value. C. LINC00467 highly expressed in most types of cancer in the GEPIA database. D. Compared with OSE and FTE, the expression of LINC00467 was higher in ovarian cancer tissues(P<0.01). E. Overall survival (OS) curve of patients with high LINC00467 expression was significantly lower than that of patients with low expression (high vs. low group: OS, HR, = 2.44; 95% confidence interval [CI] = 1.16 to 4.76 vs. HR, = 0.41; 95%CI, = 0.21 to 0.86; P = 0.02). F. Patients with platinum-resistant HGSOC maintain much higher LINC00467 expression level than those who have platinum-sensitive HGSOC (P < 0.01)

LINC00467 promotes cell proliferation and metastasis in HGSOC

A2780 cells were transiently transfected with LINC00467 siRNA, and SKOV3 cells were stably transfected with the LINC00467 overexpression vector. The efficiency of down-regulation or up-regulation was confirmed by qRT-PCR (Fig. 2A). In A2780 cells, the MTT assay demonstrated that LINC00467 down-regulation significantly decreased the relative cell viability compared with the NC group, while in SKOV3 cells, LINC00467 overexpression enhanced relative cell viability, especially in the last two days (Fig. 2B and C). In the transwell assay, Compared with the NC group, the number of invasive and imgranted cells in A280 cells with siRNA transfection decreased significantly, but the number of invasive and imgranted cells in SKOV3 cells with LINC00467 overexpression increased significantly. (Figure 2D and E). The number of cloning cells with siRNA transfection LINC00467 overexpression decreased and increased significantly respectively, (Fig. 2F and G). Furthermore, the expression of the protein marker associated with epithelial-mesenchymal transition (EMT)) (such as E-cadherin, N-cadherin, β-Catenin, and vimentin) and the cell cycle (such as CDK4 and CDK6) was greatly altered in cells with siRNA transfection or LINC00467 overexpression via western blotting, (Fig. 2H). These results indicated that LINC00467 promotes HGSOC cell proliferation and metastasis in vitro.

Fig. 2.

A. The expression of LINC00467 transfected by siRNA and LINC00467 overexpression vector in A2780 or SKOV3. B. The growth of cells transfected by siRNA in A2780. C. The growth of cells transfected by LINC00467 overexpression vector in SKOV3. D. The migration and invasion of A2780 cells transfected by siRNA in transwell assay. E. The migration and invasion of SKOV3 cells transfected by LINC00467 overexpression vector in transwell assay. F. The clonogenic capacities of A2780 cells transfected by siRNA. G. The clonogenic capacities of SKOV3 cells transfected by LINC00467 overexpression vector. H. EMT and cell cycle associated markers alteration were detected via western blotting

LINC00467 mediated PARPi resistance of HGSOC in vitro and in vivo

A2780 cells transfected with siRNA or NC were treated with different concentrations for 60 h. The MTT assay revealed that the relative cell viability of cells with LINC00467 down-regulation declined much more rapidly than that of NC cells, especially at concerntration of 40µM or 60µM (Fig. 3A). Besides, SKOV3 overexpressing LINC00467 exhibited significantly lower relative cell viability than the NC group when treated with olaparib for 48 h (Fig. 3B). Conversely, the relative cell viability of SKOV3-OR treated with olaparib with siRNA transfection was lower than the NC group (Fig. 3C). Additionally, an increase in LINC00467 expression was observed when cells were exposed to the increasing dose of olaparib (Fig. 3D). The variation trend of the cloning cells with siRNA transfection and LINC00467 overexpression was same whether treated with olaparib or not. (Figure 3E and H). Furthermore, tumor formation experiments in nude mice were designed to investigate whether elevated LINC00467 expression affected the tumor response to olaparib in vivo. Among the four groups, the tumor mass or weight comparison graph illustrated that LINC00467 elevated the tumor burden of HGSOC treated with or without olaparib in vivo (Fig. 3I and J). These assays confirmed that LINC00467 could mediate PARPi resistance in HGSOC, both in vitro and in vivo.

Fig. 3.

LINC00467 mediated PARPi resistance of HGSOC in vitro and in vivo. A.Relative cell viability of A2780 cells with or without siRNA transfection when exposed to different dose of olaparib. B. Relative cell viability of SKOV3 cells with or without LINC00467 overexpression vector transfection when exposed to different dose of olaparib. C. Relative cell viability of SKOV-OR cells with or without siRNA transfection when exposed to different dose of olaparib. D. The expression of LINC00467 in A2780 and SKOV3 cells exposed to different dose of olaparib. E-H. The colony formation efficiency of A2780 and SKOV3 cells with transfection of siRNA or LINC00467. I. The tumor mass or weight comparison graph in tumor formation experiments by BALB/c nude mice. J. the tumor burden of SKOV3 cells with olaparib-exposion and LINC00467 overexpression or not

LINC00467 could function as ceRNA via directly sponging miR-485-5p

The subcellular fractionation experiment revealed that LINC00467 was mainly located in the cytoplasm of A2780 and SKOV3 cells compared with MALAT1, the positive nuclear control (Fig. 4A and B). According to available public databases, miR-485-5p was predicted to be a potential sponging target of LINC00467, which maintains one binding site (Fig. 4C). To confirm whether LINC00467 directly interacted with miR-485-5p, a dual-luciferase reporter assay was performed. As shown in Fig. 4C, miR-485-5p mimics significantly reduced luciferase activity in HEK293T cells transfected with LINC00467 WT pmirGLO reporter vector, but not in cells transfected with the LINC00467 MT reporter vector (Fig. 4C). Furthermore, the relative expression levels of LINC00467 and miR-485-5p in our HGSOC cohort were measured, and it was concluded that miR-485-5p levels were negatively correlated with LINC00467 (Pearson r=−0.3769, P < 0.01) (Fig. 4D), which was similar to the trend observed in HGSOC cell lines (Fig. 4G).

Fig. 4.

A. and B. The ratio of nuclear expression to cytoplasmic expression of LINC00467 in subcellular fractionation, compared to MALAT1 and APT. C. The association between luciferase activity of cells transfected by LINC00467 WT(MT) pmirGLO reporter and miR-485-5p mimics in dual-luciferase reporter. D. Relative miR-485-5p level was negatively correlated with LINC00467 in tumor samples (Pearson r, =−0.3769; P < 0.01). E. The association between luciferase activity of cells transfected by RAD50 3’UTR WT(MT) pmirGLO reporter and miR-485-5p mimics in dual-luciferase reporter. F. Relative RAD50 level was negatively correlated with miR-485-5p in tumor samples (Pearson r, =−0.4638; P < 0.01). G. miRNA-485-5p was regulated by LINC00467 in A2780 and SKOV3. H. Western blotting illustrated the regulation of miR-485-5p in RAD50 protein expression

RAD50 was the direct target of miR-485-5p

Based on the results of TargetScan 7.1, Rad50 is a putative target gene of miR-485-5p. One potential binding site was found in the 3’-UTR of RAD50 (Fig. 4E). The luciferase reporter assay confirmed that miR-485-5p mimics significantly decreased the luciferase activity of the RAD50 WT reporter vector, but no considerable change was observed in the RAD50 MT reporter vector (Fig. 4E). A negative correlation between the expression of miR-485-5p and RAD50 was confirmed in this cohort (Pearson r=−0.4638, P < 0.001) (Fig. 4F). Accordingly, the protein level of RAD50 was enhanced by the miR-485-5p inhibitor but impaired by miR-485-5p mimics in A2780 and SKOV3 cells, as determined by western blotting (Fig. 4H).

LINC00467 mediated PARPi resistance via miR-485-5p/RAD50 axis

Furthermore, we designed a rescue experiment by transiently transfecting miR-485-5p mimics or inhibitors into A2780 or SKOV3 cells with siRNA or a LINC00467 overexpression vector. In gradient concentrations of olaparib, the relative cell viability was measured, which revealed that the miR-485 inhibitor could partly abrogate the PARPi-sensitizing effect of siRNA, while miR-485 mimics could enhance the response to PARPi in LINC00467 overexpressing SKOV3 (Fig. 5A and B). Furthermore, miR-485-5p expression levels were also altered by the transfection of mimics or inhibitor (Fig. 5C and D), in contrast to the response to olaparib. RAD50 expression was positively regulated by LINC00467, but negatively regulated by its siRNA, which could be rescued by transfection with miR-485-5p mimics or inhibitor (Fig. 5E and F). In the nude mouse tumor experiments, we also detected the expression levels of RAD50 in tumor tissues of different groups of nude mice. The expression level of RAD50 in the LINC00467 overexpression group was significantly higher than that in the NC group both in DSMO condition and Oraparib condition (Fig. 5G and H). This experiment clearly demonstrated from an in vivo perspective that LINC00467 and RAD50 are involved in PARPi resistance in HGSOC. These assays demonstrated that LINC00467 mediated PARPi resistance in HGSOC via the miR-485-5p/RAD50 axis.

Fig. 5.

A and B. The association between a dose of olaparib and cell viability in A2780 and SKOV3, which is transfected by siRNA or miR-485-5p inhibitor or mimics. C and D. qRT-PCR assay revealed the relative expression of miR-485-5p in different treated cells. E and F. RAD50 protein expression was detected by western blotting. The expression alteration of RAD50 was rescued by miR-485 mimics or inhibitor transfection.G. Comparison graphs of tumor tissues for RAD50 in BALB/c nude mouse tumor formation experiments. H. Data analysis of tumor tissues for RAD50 in L nude mouse tumor experiments

LINC00467 ASO played a synergistic role with olaparib in HGSOC PDX models

To further investigate the therapeutic effect of LINC00467 in patient-derived tumors, we established two HGSOC PDX cases and synthesized LINC00467 ASO for treatment. Each patient was divided into an olaparib/DDW group (n = 3) and an olaparib/ASO group (n = 3). After abdominal injection twice a day for 4 weeks, the tumor burden of the olaparib/ASO group that received the synergistic regimen of olaparib and LINC00467 ASO was significantly lower than that of the single olaparib group in both PDX cases (Fig. 6A and B). The abdominal metastasis nodes were indicated by red arrows and compared (Fig. 6A). In detail, the abdominal metastasis in the olaparib/ASO reduced significantly compared to olaparib/DDW (Fig. 6C and D). Additionally, the expression of RAD50 was lower in the olaparib/ASO than olaparib/DDW(Fig. 6E). We conducted immunofluorescence assays for RAD50 and γH2AX on tumor tissue sections of the PDX model. As depicted in the figure, compared with the olaparib/saline group, the proportion of RAD50 - positive and γH2AX - positive cells in the olaparib/ASO group was significantly lower (p < 0.001). Similarly, the pattern of γH2AX - positive cells also showed a significant decrease (p < 0.001) (Fig. 6G - H).These findings suggest that the olaparib/ASO group exhibits a relatively poor DNA damage response and repair capacity. This compellingly demonstrates that the low expression of LINC00467 leads to a reduction in HRR expression, providing conclusive evidence that LINC00467 mediates PARPi resistance in HGSOC. In summary, LINC00467 sponged miR-485 to upregulate RAD50 expression, which contributed to PARPi resistance in HGSOC (Fig. 6F).

Fig. 6.

LINC00467 improved response to olaparib in HGSOC PDX cases. A. Two cases were established and treated with olaparib singly or combination of olaparib and ASO. B. The tumor burden of the mice treated with Olaparib/ASO and olaparib/DDW group. C and D. The abdominal metastasis number of two PDX cases between Olaparib/ASO and olaparib/DDW group E. The comparison of RAD50 mRNA expression level between different groups of mices treated with Olaparib/ASO and olaparib/DDW. F. Graphical abstract of this study showed that LINC00467 could sponge miR-485 to restore the RAD50 expression and HR repair pathway, which caused PARP inhibitor resistance. G. Immunofluorescence expression images of RAD50 (red) and γ-H2AX (orange) in PDX model tumor tissues. H. Immunofluorescence statistical test graphs of RAD50 and γ-H2AX in PDX model tumor tissues

Discussion

Despite the continuous improvement in living standards, surgery and chemotherapy in recent years, the overall survival rate of ovarian cancer has not improved and the 5-year survival rate is only approximately 30% [3, 18]. PARPi is currently the most promising targeted therapy for epithelial ovarian cancer [19] based on its synthetic lethal effect [20]. Considering the results of the SOLO1 phase III clinical trial, the U.S. FDA approved olaparib as a first-line maintenance therapy for patients with advanced-stage epithelial ovarian cancer with deleterious or suspected deleterious germline or somatic BRCAm (gBRCAm or sBRCAm) [21]. Nevertheless, 40–70% of patients with BRCAm are still resistant to PAPRi therapy [8, 9, 22], indicating that BRCAm detection alone is not an ideal predictor. In response to this situation, some international scholars have developed HRD detection methods to predict the sensitivity and prognosis of patients with PARPi, mainly including the HRR gene panel [23] and the HRD genomic scar [24, 25]. Several studies have shown that sensitivity to chemotherapy, PARPi, and prognosis of HRD-positive patients are significantly better than those of HRD-negative patients. However, some HRD-negative patients may benefit from PARPi. Thus, we induced a PARPi-resistant HGSOC cell line and performed lncRNA sequencing to obtain the PARPi response-associated lncRNA——LINC00467.

Recent studies have demonstrated that lncRNAs play important roles in various physiological and pathological processes [26, 27]. In recent years, many lncRNAs have been identified in mammalian transcriptomes [28]. LINC00467 is an important oncogene in several types of cancer. Ge et al. illustrated that LINC00467 could encode the micropeptide ASAP and promote colorectal cancer progression by directly modulating ATP synthase activity [29]. Zhu et al. reported that LINC00467 is highly expressed in NSCLC tissues and associated with advanced clinical stages and poor outcomes. LINC00467 expression is regulated by TDG-mediated acetylation and promotes NSCLC progression via the Akt signalling pathway [30]. Ji et al. proved LINC00467 stimulates proliferation, migration and invasion capacities of melanoma via targeting miR-485-5p to upregulate PAK1, which provides potential targets for treatment of melanoma [31]. Liang et al. reported that Linc00467 can be used as ceRNA to adsorb miR-1285-3p to regulate the expression of TFAP2A, promote invasion and inhibit apoptosis of head and neck squamous cell carcinoma(HNSCC) cells. Linc00467 inhibitors may become one of the targeted therapeutic drugs for HNSCC [32]. Liu et al. proved that LINC00467 acted as an oncogene in Esophageal squamous cell carcinoma by accelerating cell proliferation and preventing cell apoptosis via miR-485‐5p/DPAGT1 axis [33]. However, the effect of LINC00467 on HGSOC tumorigenesis has not been elucidated. In this study, Through database prediction and multiple verifications including dual-luciferase reporter gene assays, we first identified LINC00467 as an important differentially expressed gene between PARPi-resistant cells and original cells using lncRNA sequencing and bioinformatics analysis. It was further proven to promote HGSOC proliferation and metastasis in cell biology experiments. In vivo and in vitro assays also confirmed that LINC00467 could impair the response to PARPi in HGSOC cells.

Abnormal cellular responses to DNA damage are pivotal in tumorigenesis. Cells employ diverse DNA repair pathways to rectify different forms of DNA lesions, including base excision repair, mismatch repair, nucleotide excision repair, direct repair, and recombinational repair, such as homologous recombination repair (HRR) and non-homologous end joining (NHEJ). HRR is a highly intricate process involving multiple key genes, including ATM/ATR, CHK2/CHK1, BRCA1/2, H2AX, MRE11, RAD50, and RAD51 [20]. Previous studies have indicated that miR-485-5p can suppress ovarian cancer progression by targeting SRC [34]. Li et al. demonstrated that RAD50 enhances ovarian cancer progression through NF-κB activation [35]. RAD50 deficiency has also been identified as a predictor of platinum sensitivity in sporadic epithelial ovarian cancers [36].

Moreover, Zetrini et al. established that silencing RAD50 via RAD50-siRNA nanoparticles can impair radiation-induced DNA damage repair, significantly enhancing the efficacy of radiotherapy in triple-negative breast cancer [37]. In the present study, we show, for the first time, that miR-485-5p interacts with LINC00467 and directly targets RAD50, a critical gene within the HRR pathway. By disrupting HRR, LINC00467 contributes to PARP inhibitor resistance, mirroring the “synthetic lethality” effect observed in other cancers.Despite LINC00467 being implicated in promoting tumorigenesis across several malignancies—such as lung adenocarcinoma, gastric cancer, colorectal cancer, hepatocellular carcinoma, breast cancer, osteosarcoma, and head and neck cancers [38]. —its mechanism of action generally involves acting as a “miRNA sponge,” thereby upregulating oncogenic pathways. In esophageal squamous cell carcinoma, for example, LINC00467 modulates the miR-485-5p/DPAGT1 axis [39]. Conversely, miR-485-5p has been recognized for its tumor-suppressive properties in various cancers, including gastric cancer, breast cancer, cervical cancer, melanoma, and lung cancer [40].

The novelty of this study lies in two pivotal findings: First, we are the first to identify the LINC00467/miR-485-5p/RAD50 axis as a key regulatory pathway in high-grade serous ovarian cancer (HGSOC). Second, we demonstrate the specific significance of this axis in HGSOC: RAD50, a gene frequently overexpressed in HGSOC, is strongly associated with poor prognosis, with elevated RAD50 expression contributing to tumor invasion and cell proliferation [40]. While the LINC00467/miR-485-5p axis has been previously described in other cancers, its association with RAD50 has not been explored in the context of HGSOC, which is notably characterized by homologous recombination deficiency (HRD). Indeed, one of the defining features of HGSOC is HRD, making RAD50—a key player in the DNA damage response—particularly relevant in this malignancy [41].

Thus, our findings not only unveil a novel oncogenic mechanism involving LINC00467 in HGSOC but also highlight the potential of the LINC00467/miR-485-5p/RAD50 axis as a distinct molecular signature or therapeutic target for HGSOC. Unlike other cancers where LINC00467 predominantly exerts a “universal” oncogenic role, our study underscores the mechanistic specificity of this axis in HGSOC, particularly through its interaction with RAD50 in the context of DNA damage repair. From a clinical standpoint, HGSOC’s sensitivity to DNA damage repair pathways (e.g., BRCA1/2, RAD51-like genes) and treatment strategies, such as PARP inhibitors and platinum-based chemotherapy, underscores the potential clinical significance of targeting the LINC00467/miR-485-5p/RAD50 axis in modulating treatment responses and overcoming resistance mechanisms.However, we acknowledge that, to date, no studies have definitively demonstrated the existence of the LINC00467/miR-485-5p/RAD50 axis in other cancers. This gap underscores the contribution of our work in filling this void. Future studies should explore whether this axis exists or exhibits distinct effects in other cancer types, further validating its specificity to HGSOC.

Most traditional treatment studies on ovarian cancer have used established immortalized cell lines, which ignore the common heterogeneity of HGSOC. The PDX model is an advanced in vivo model for tumor research. Human tumor specimens were collected into immune-deficient mice for passaging and a series of experimental studies without additional in vitro intervention [42]. Compared with traditional cell line tumorigenesis, the PDX model is established based on an individual patient, which can not only better simulate the behavioral characteristics of the primary tumor, but is also an ideal model for individualized treatment research [43, 44]. Some scholars have successfully constructed a subcutaneously transplanted PDX model of ovarian cancer, as well as intraperitoneal planted models that effectively simulate in vivo metastasis [45, 46]. In our study, LINC00467 ASO was synthesized and used in HGSOC PDX models to further investigate whether lncRNA therapy can be applied to human tumor tissues. In our results, we also observed a significant increase in the proportion of RAD50-positive and γH2AX-positive cells in the LINC00467 overexpression group, which strongly demonstrate that upregulation of RAD50 was related to the number of γH2AX foci increasing, suggesting that overexpression of RAD50 may disrupt DNA damage repair processes and contribute to increased resistance to PARPi. Previous studies have shown that RAD50, as a key component of the MRN complex, is crucial for DNA damage sensing and repair [47]. However, excessive expression of RAD50 may negatively regulate the homologous recombination repair (HRR) pathway, leading to an accumulation of γH2AX markers of damage [48] This disruption in repair capacity enhances cellular tolerance to PARP inhibition, thereby promoting drug resistance. Therefore, LINC00467-mediated upregulation of RAD50 likely plays a significant role in PARP inhibitor resistance, further reinforcing our findings. With the help of two enrolled PDX cases, we confirmed that combination therapy with LINC00467 and olaparib could significantly depress tumor growth along with a decrease in RAD50 expression, which brings new possibilities for lncRNA therapy in HGSOC to some extent.

However, this study has the following limitations: Although it preliminarily reveals the potential mechanism by which LINC00467 influences tumor cell resistance phenotypes through the regulation of RAD50 expression, certain limitations remain. This research confirms a significant correlation among LINC00467 overexpression, high RAD50 expression, and tumor cell resistance phenotypes. Additionally, transcriptional regulatory experiments have validated the regulatory effect of LINC00467 on RAD50; however, direct evidence confirming that RAD50 is a key downstream effector molecule mediating the resistance phenotype induced by LINC00467 was not obtained through rescue experiments involving RAD50 knockdown. Therefore, it is currently impossible to completely rule out the possibility of other intermediate regulatory factors existing between them. While RAD50 may be an important component within the regulatory network modulated by LINC00467, it is not necessarily the sole mediator. Future studies should conduct rescue experiments using siRNA/CRISPR-mediated knockdown of RAD50 in stable cell lines overexpressing LINC00467 to verify whether knocking down RAD50 can reverse its resistance phenotype. This would enhance both the rigor and translational value of our conclusions. It is undeniable that the PDX model designed in this study (n = 3) has a small sample size that makes it difficult to entirely eliminate individual differences affecting experimental results. In subsequent research, efforts should be made to expand both PDX model sample sizes and clinical sample data while further exploring correlations between tumor heterogeneity and treatment efficacy. Such improvements will contribute to refining research content and enhancing both reliability and clinical applicability of our findings.

Conclusions

In our study, we identified a carcinogenic role of LINC00467 in HGSOC. The overexpression of LINC00467 significantly promoted cell proliferation, migration, and invasion. LINC00467 impairs the response to PARPi in vitro and in vivo. In addition, we identified that LINC00467 could function as a ceRNA against miR-485-5p, which further regulates RAD50 and impacts the HRR process (Fig. 6F). The newly identified LINC00467/miR-485-5p/RAD50 axis provides novel insights into PARPi resistance in HGSOC and represents a potential therapeutic target for the clinical treatment of HGSOC.

Authors’ contributions

Xin Li1,#, Yi Xiang2,#, Jie Gao1, Hui Zong1, Miao Li1, Renzhuo Gao1 and Hui Zhang1*#Xin Li and Yi Xiang contributed equally to this work* Correspondence: Hui ZhangHui Zhang: funding acquisition, project administration, supervision. Xin Li: visualization, methodology, writing-original draft. Yi Xiang: visualization, methodology, writing-original draft. Jie Gao, Hui Zong: formal analysis, data curation. Miao Li, Renzhuo Gao: conceptualization, software.

Funding

This study was supported by Shandong Province Natural Science Foundation (ZR2020MH273), and Key Research and Development Program of Shandong Province, China (2022CXGC010504-3).

Data availability

The data used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.