KDM5D expression is lost in cisplatin‑resistant neuroblastoma cells

Introduction

Covalent histone methylation of lysine residues carries out a key role in regulating chromatin dynamics and functions (1–3). Methylation of lysines on histone H3 and H4 activates or represses gene transcription, depending on the position of the modified residues (1,4). In general, methylated histone H3 lysine 4 (H3K4) is associated with active or balanced gene states, whereas methylated H3K9 and H3K27 are gene repressive (5). The removal of methyl groups from lysine residues on histones is regulated by histone lysine demethylases (KDM) (6,7).

To date, two distinct families of demethylases have been described the flavin-dependent KDM1 and the JmjC domain-containing KDM2-8 subfamilies. The first family, KDM1, catalyzes the demethylation of mono- and di-methylated lysine residues (Kme1 and Kme2). The second family of KDMs (KDM2-8) is capable of demethylating Kme1, Kme2 and Kme3 (8). In addition, KDMs demethylate non-histone substrates and also have several demethylase-independent functions (9). The KDM expression profile varies in different cells and tissues. Altered expression of KDMs, especially those targeting H3K4 and H3K27, is common in several types of human cancer (9). The KDM5 subfamily, that has 4 members (KDM5A-D), is capable of removing tri- and di- methyl marks from H3K4 (6). This subfamily is deregulated in several types of cancer and can modulate chemoresistance by numerous mechanisms including autophagy, epithelial-mesenchymal transition (EMT), stemness, metabolism and DNA repair (6,9). The role of KDM5s in the development of chemoresistance has been described in various types of cancer (10) KDM5A (11–14); KDM5B (15–21) and KDM5C (22–24).

The KDM5D gene (also known as JARID1D or SMCY) is located on the Y chromosome and it is the only male-specific KDM5. It is expressed in all male tissues, and carries out a role in spermatogenesis (25,26). KDM5D has been described as an important tumor-suppressor in castration-resistant prostate cancer and its low expression is associated with a worse prognosis (27). In prostate cancer, KDM5D regulates matrix metalloproteinase family genes associated with invasion, and loss of KDM5D with increased H3K4me3 levels in promoter regions of relevant genes increases invasiveness and metastatic ability (28,29). Moreover, in clear cell renal cell carcinoma (ccRCC), KDM5D is downregulated by loss of the Y chromosome, which contributes to the pathogenesis of ccRCC (30). In papillary renal cell carcinoma, KDM5D also facilitates demethylation of CDK4 and promotes proliferation of cancer cells (31). Chen et al (32) described the function of KDM5D in the development of CDDP tolerance in head and neck squamous cell carcinoma (33). Decreased KDM5D expression was also observed in gastric, colorectal and hepatocellular carcinomas (34–36). Moreover, in an analysis of the Cancer Genome Atlas database, Duan et al (37) showed that KDM5D was notably downregulated in 24 different cancer types (such as breast, pancreatic and prostate cancer) compared with adjacent tissues. To the best of our knowledge, little is known about the importance of KDM5D in neuroblastoma. However, it has been reported that low KDM5D expression is associated with a worse prognosis in some tumors (38,39). We hypothesize that this is due to the decrease of cell junctions and therefore increase the ability to metastasize and due to the decrease of the presentation of antigens and therefore the immune response to the tumor (38,39).

CUL4A is a protein of the cullin family that acts as a scaffold for cullin RING ligase 4 complexes that promote ubiquitination of various substrates. It carries out an important role in DNA repair and replication, chromatin restructuring, cell cycle regulation, embryogenesis, hematopoiesis and spermatogenesis (40,41). There are a growing number of studies associating overexpression or amplification of CUL4A to increased growth, progression and metastasis in cancer (42–53). The CUL4A gene is located on 13q34, an area prone to amplification in some types of cancer (45–53). Shen et al (34) demonstrated that KDM5D carries out an important role in the induction of EMT of gastric cancer cells through demethylation in the promoter of CUL4A in male patients. In addition, another study suggests a relationship between the CUL4A gene and the sensitivity of colorectal cancer cells to CDDP (53).

Neuroblastoma is a malignancy of the sympathetic nervous system and is the most common malignant extracranial tumor of childhood. It is characterized by high degree of heterogeneity, which may account for the wide range of clinical presentations and variable response to treatment (54). The combination of clinical and genetic factors allows stratification of patients into very low, low, intermediate and high-risk groups (55). High-risk neuroblastoma is characterized by the development of acquired chemoresistance (56). To the best of our knowledge, there is currently little information regarding the role of KDM5D in neuroblastoma and the contribution of KDM5D to CDDP-chemoresistance.

The present study aims to investigate the importance of KDM5D expression for the proliferation of neuroblastoma cells and their chemoresistance to CDDP. On the basis of the aforementioned studies that demonstrated an association between KDM5D and CUL4A and their contribution to chemoresistance to CDDP, the present study investigated whether changes of CUL4A expression could be mediated by KDM5D in neuroblastoma cell lines.

Materials and methods

Cell culture and chemicals

Human high-risk neuroblastoma cell lines UKF-NB-3 and the derived CDDP-resistant line UKF-NB-3CDDP were provided by Prof. Jindrich Cinatl, Goethe University, Frankfurt am Main, Germany. IMR-32 was purchased from MillporeSigma and SK-N-F1 by American Type Culture Collection. The CDDP-chemoresistant cell lines IMR-32CDDP and SK-N-F1CDDP were derived in our laboratory from their chemosensitive parental cell lines (IMR-32 and SK-N-F1) after long-term cultivation with increasing CDDP (Sandoz Group AG) concentration (57,58). All tested cell lines were of male origin. Cells were cultured at 37°C and 5% CO2 in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% (v/v) fetal bovine serum (both Thermo Fisher Scientific, Inc.). KDOAM-25 citrate (400 nM; HY-102047B; MedChemExpress) was used to inhibit KDM5s, where it was added to cells and incubated for 48 h at 37°C.

KDM5 inhibition using KDOAM-25

For the inhibition of KDM5 demethylases, cells were treated with KDOAM-25 citrate for 24–48 h at 37°C and 5% CO2 (cat. no. HY-102047B; MedChemExpress) at a final concentration of 400 nM. This concentration was selected based on preliminary optimization experiments demonstrating efficient reduction of H3K4 trimethylation without affecting cell viability (data not shown). For supplementary validation experiments, KDOAM-25 was added 24 h before CDDP treatment, whereas for all main experiments presented in the manuscript, KDOAM-25 was added 48 h before CDDP administration and maintained throughout the treatment period.

Cell proliferation assay

To determine cell proliferation, cells were placed in 24-well plates (1×105 cells per well) or 96-well plates (1×104 cells per well) and seeded for 24 h at 37°C.and then cells were treated with CDDP at a final concentration of 0.6-300 µM for 48 h at 37°C. Cells were then incubated with PrestoBlue® Cell Viability Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Fluorescence was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm using the SpectraMax® i3× Multi-Mode Microplate Reader (Molecular Devices, LLC). Each sample was analyzed in triplicate. The optical density of the medium was read as background, and the value of the optical density of the control cells was taken as 100%. IC50 values were calculated using SOFTmax® Pro 7.2 GxP software (Agilent Technologies, Inc.).

Transfection

On-Targetplus small interfering (si)RNA (Revvity Discovery Limited), a smart pool of three siRNAs [12.5 or 25 nM; cat. no. L-007948-00-0005; National Center for Biotechnology Information (NCBI) accession nos. NM_001146705.2, NM_001146706.2 and NM_004653.5] was used for silencing of KDM5D and On-Targetplus Non-Targeting control siRNA (12.5 or 25 nM; cat. no. D-001320-01-20), a smart pool of four siRNAs, was used as a negative control (Revvity Discovery Limited). For initial validation experiments, siRNA was tested at 12.5 and 25 nM. Based on these results, a final concentration of 25 nM was used for all subsequent functional experiments. siRNAs were transfected with Dharmafect transfection reagent (cat. no. T-2001-03; Revvity Discovery Limited) for 48 h according to the manufacturer's protocol. Following 48 h of incubation at 37°C, cells were harvested and used for further analysis.

To ectopically express KDM5D, UKF-NB-3CDDP cells were transfected for 48 h with the GenEZTM ORF clone plasmid KDM5D pCMV-3Tag1a (OHu18895C; Genscript) designed for transcript variant 1 of KDM5D (8 and 16 ng/µl; NCBI accession number NM_001146705.1; GenScript Biotech Corporation) and pCMV6-AC-GFP, mammalian vector with C-terminal tGFP tag (20 nM; OriGene Technologies, Inc.) was used as a negative control. For initial validation experiments, siRNA was tested at 8 and 16 ng/µl. Based on these results, a final concentration of 16 ng/µl was used for all subsequent functional experiments. The KDM5D ORF clone plasmid or control pCMV6-AC-GFP was transfected using Dharmafect transfection reagent (T-2001-03; Revvity Discovery Limited). The transfected cells were selected by Gibco geneticin™ Selective Antibiotic (cat. no. G418 Sulfate; 50 mg/ml) (Thermo Fisher Scientific, Inc.) in a final concentration 400 µg/ml for 72 h at 37°C. Overexpression efficiency was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and flow cytometry respectively.

The exact siRNA/shRNA sequences used in the present study are proprietary to Revvity Discovery Limited and are not publicly available. They can be obtained for manufacturer upon reasonable request (www.dharmacon.com).

RT-qPCR

mRNA from all NBL cell lines was isolated using the PureLink™ RNA Mini Kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The quality of the extracted mRNA was measured using the NanoDrop One spectrophotometer (260/280 nm ratio) (Thermo Fisher Scientific, Inc.). Complementary DNA was synthesized from 1.0 µg of mRNA using the Generi Biotech Reverse Transcription Kit according to the manufacturer's instructions (GENERI BIOTECH). RT-qPCR was performed using the gb Easy PCR Master Mix (cat. no. 3006; GENERI BIOTECH) according to the manufacturer's instructions, with custom primers (Generi custom oligo synthesis) and hydrolysis probes. The RT-qPCR assays are identified by Generi Biotech IDs. These assays can be ordered at ID (ID: ‘hKDM5B_Q1’ and ‘hKDM5D_Q1’) from GENERI BIOTECH. Expression levels of target genes and the internal control POLR2A (ID, hPOLR2A ‘hPOLR2A_Q1’), which is homogeneously and uniformly expressed in neuroblastoma cell lines (59), were analyzed by RT-qPCR on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Inc.). Each sample was analyzed in triplicate. The thermocycling conditions were: 95°C 3 min, 50 cycles of 95°C for 10 sec, 60°C 20 sec. The relative differences in gene expression were expressed as fold change and were obtained with the 2−ΔΔCq method (Relative Expression Software Tool REST 2009 software, v2.0.11; QIAGEN) (60). The exact primer sequences used in the present study are proprietary to GENERI BIOTECH and are not publicly available. They can be obtained for manufacturer upon reasonable request (www.generi-biotech.com).

Cell cycle analysis

Cell cycle analysis was carried out using FxCycleTM Violet Ready FlowTM reagent (Thermo Fisher Scientific, Inc.). After treatment with CDDP and/or transfection, harvested neuroblastoma cells with 0.25% trypsin (Thermo Fisher Scientific, Inc.) were pelleted by centrifugation (300 × g) for 3 min at room temperature, resuspended in 100 µl of 3.6% paraformaldehyde (Biogen) and incubated at 20°C for 15 min. After this incubation, the suspension was then centrifuged (300 × g) for 3 min at room temperature, and the pellet was washed twice with phosphate-buffered saline (PBS; Thermo Fisher Scientific, Inc.). Permeabilization was performed with 90% methanol (PENTA) for 1 h at −20°C. Pellets were washed with PBS, resuspended in 500 µl of PBS and a drop of FxCycleTM Violet Ready FlowTM reagent was added. After 30 min of incubation at room temperature, cell cycle analysis was performed using a BD FACSCelesta (BD Biosciences) and data were analyzed using Flowlogic software, version 8 (Inivai Technologies).

Determination of protein levels and histone H3K4 methylation status by flow cytometry

After treatment with CDDP and/or transfection, harvested neuroblastoma cells (UKF-NB-3; UKF-NB-3CDDP) were washed with cold PBS (Thermo Fisher Scientific, Inc.), trypsinized with 0.25% trypsin (Thermo Fisher Scientific, Inc.) and collected by centrifugation (300 × g) for 3 min at room temperature. Cell pellets were washed with PBS and fixed in 3.6% paraformaldehyde for 15 min at room temperature. The cell pellets were then washed with PBS and permeabilized with 90% methanol for 1 h at −20°C. The pellets were then washed three times with 0.5% bovine serum albumin (BSA; Roth) in PBS. After washing, the cell pellets were blocked with 5% BSA in PBS for 1 hour at room temperature After blocking, the pellets were incubated with the primary antibody anti-JARID1D rabbit mAB at a dilution of 1:100 (cat. no. PA5-100844; Invitrogen, Thermo Fisher Scientific, Inc.), anti-JARID1B rabbit mAB at dilution of 1:400 (cat. no. 15327S; Cell Signaling Technology, Inc.), anti-trimethyl histone H3 (Lys4) rabbit (cat. no. 07-473; MilliporeSigma) at a dilution of 1:400, Cleaved-Caspase 3 (Asp175) Rabbit mAB at a dilution of 1:50 (cat. no. 9602S; Cell Signaling Technology, Inc.) or CUL4A Rabbit mAB at a dilution of 1:50 (cat. no 2699; Cell Signaling Technology, Inc.) for 1 h at laboratory temperature. Cell pellets were then washed with 0.5% BSA (Roth) and incubated in fluorochrome-conjugated secondary antibody Anti-Rabbit IgG (H+L) Alexa Fluor® 647 Conjugate (cat. no. A21245; Thermo Fisher Scientific, Inc.) diluted 1:500 and incubated for 30 min at room temperature in the dark. Cell pellets incubated with secondary antibody (1:500) only were used as a control. Washed and resuspended cells were measured using a BD FACSCelesta (BD Biosciences), and data were analyzed using Flowlogic software, version 8 (Inivai Technologies).

Cell migration monitoring

Real-time monitoring of cell migration of sensitive cells (UKF-NB-3) and their derived chemo-resistant cells (UKF-NB-3CDDP) was carried out using the xCELLigence RTCA DP instrument (Agilent Technologies, Inc.) in a humidified incubator at 37°C and 5% CO2. For sensitive cells, KDM5D silencing (siKDM5D) and for CDDP-resistant UKF-NB-3CDDP ORD cDNA transfection were performed. Cells were serum-starved for 2 h in IMDM without FBS at 37°C and 5% CO2. After the starvation period cells were trypsinized and seeded at a density of 1×104 cells/well into upper chamber of the 16-well electronically integrated Boyden chamber for invasion/migration assays-CIM-plate 16 (RTCA, Agilent) containing starvation media. The wells of the lower chamber were loaded with IMDM with 10% FBS. As the cells migrated towards the chemoattractant across microelectronics sensors integrated at the bottom side of upper chamber, the impedance was measured every 30 min for 168 h at 37°C. The measurements were recorded and analyzed using Real-Time Cell Analysis Software 1.2 (Agilent Technologies, Inc.).

Cell proliferation monitoring

Real-time monitoring of cell proliferation was carried out using the xCELLigence RTCA DP instrument (Agilent Technologies, Inc.) in a humidified incubator at 37°C with 5% CO2. Sensitive cells and their derived chemoresistant cells (UKF-NB-3, UKF-NB-3CDDP, IMR-32, IMR-32CDDP, SK-N-F1 or SK-N-F1CDDP) were seeded at a concentration 8,000 cells per well in wells of 16-well E plates for impedance-based detection. For sensitive cells KDM5D silencing (siKDM5D) was carried out. After 48 h of transfection (siKDM5D), cells were seeded at a concentration 8,000 cells per well in wells of 16-well E plates for impedance-based detection. The cell index was monitored every 30 min for 96 h at 37°C and data were recorded using the xCELLigence RTCA software Pro version 2.8 (Agilent Technologies Inc.) provided.

R2 genomics analysis and visualization platform

The R2 Genomics Platform (https://r2.amc.nl) is an open-access online genomic analysis and visualization platform that is publicly available to analyze and interpret clinical and genomic data. Several neuroblastoma datasets are available for survival analysis. The present study used three different datasets: i) Tumor neuroblastoma from Kocak (649; custom; ID: Agilent 44K microarray, Kocak dataset, Wolf normalization-ag44kcwolf;), which contains gene expression profiles from 649 patient-derived neuroblastoma tumors (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi R2 internal identifier: ps_avgpres_gse45547geo649_ag44kcwolf) (61); ii) tumor neuroblastoma from SEQC (498; RPM; ID: SEQC/MAQC-III neuroblastoma dataset - seqcnb1), which contains expression data from gene expression microarrays for 498 patient-derived neuroblastoma tumors (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi R2 internal identifier: ps_avgpres_gse62564geo498_seqcnb1) (62) and iii) tumor neuroblastoma from Oberthuer (251; user-defined; ID: Oberthuer neuroblastoma gene-expression dataset - amexp255), which consists of gene expression profiles from 251 patient-derived neuroblastoma tumors (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi R2 internal identifier: ps_avgpres_nb251_amexp255) (63), (Table SI). To analyze the prognostic significance of the KDM5D and CUL4A genes Kaplan-Meier curves were generated comparing overall survival of patients with low and high expression of KDM5D or CUL4A using the integrated plotting function with default settings on R2: Genomics Analysis and Visualization Platform (Department of Oncogenomics, Amsterdam University Medical Centers (AMC), University of Amsterdam; accessed in 2024). Median expression cutoff mode and Bon-Ferroni correction for multiple testing was performed for all analyses. Statistical significance of survival differences was determined using the log-rank (Mantel-Cox) test.

Western blot analysis

Proteins were extracted from cultured neuroblastoma cells. Extraction of proteins was conducted using the ReadyPrep™ Protein Extraction Kit (Bio-Rad Laboratories, Inc.), with the addition of a complete protease inhibitor cocktail (Roche Applied Science). The concentration of proteins was subsequently measured using a BCA Protein Assay kit (Thermo Fisher Scientific Inc.). Samples (40 µg) were resolved on 4–20% precast gradient SDS-polyacrylamide gels (Mini-PROTEAN® TGX™, Bio-Rad Laboratories, Inc.), transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.) and blotted on PVDF membranes (Bio-Rad Laboratories, Inc.). Membranes were blocked in 5% BSA in TBS containing 0.1% Tween-20 for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. The following primary antibodies were used: KDM5D Rabbit pAb (cat. no. PA5-100844; Thermo Fisher Scientific, Inc.), was diluted to a concentration of 1:500, while CUL4A Rabbit pAb (cat. no. 2699S; Cell Signaling Technology Inc.) was diluted to 1:1,000. The PARP Rabbit pAb (cat. no. 9542S; Cell Signaling Technology Inc.) and the Caspase-3 Rabbit pAb (cat. no. 9622S; Cell Signaling Technology Inc.) were both diluted to 1:1,000. β-tubulin Mouse mAb (cat. no. 86298S, Cell Signaling Technology Inc.) was diluted to 1:1,000 and used as a loading control. Secondary antibodies, AlexaFluor 488 Mouse, Rabbit (cat. nos. A-11008 and A28175; Thermo Fisher Scientific Inc.) were diluted 1:2,000 and the incubation conditions were 1 h at room temperature. The membranes were then subjected to visualization via the ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc.). The analysis was conducted utilizing ImageJ 1.52a software (National Institutes of Health).

For the verification of sustained KDM5D silencing, UKF-NB-3 cells were transfected with ON-TARGETplus KDM5D siRNA as described above and lysed at multiple time points up to 7 days after transfection. KDM5D protein levels were analyzed by western blotting using the KDM5D antibody and β-tubulin as a loading control.

Statistical analysis

All experiments were repeated independently at least three times, and data are expressed as mean ± standard error of the mean. ANOVA with post hoc Tukey honestly significant difference was used to compare different groups. One-way ANOVA with Tukey's post hoc test was used when a single independent variable (such as treatment, transfection or time) was analyzed. Two-way ANOVA with Tukey's post hoc test was applied when two independent variables (such as cell line and treatment) were analyzed simultaneously. Differences were considered statistically significant when. The exact statistical tests used for each analysis are indicated in the respective figure legends.

Results

Loss of KDM5D expression in CDDP resistant neuroblastoma cell line

Chemotherapy is one of the main treatment modalities for high-risk neuroblastoma, and CDDP is part of the majority of treatment protocols. The occurrence of chemoresistance is a considerable negative sign and contributes fundamentally to treatment failure. In this context, cell lines with intrinsic induced resistance to CDDP were analyzed. The IC50 of the CDDP-resistant cell lines (UKF-NB-3CDDP, IMR-32CDDP and SK-N-F1CDDP) was significantly higher compared with the drug-sensitive parental lines UKF-NB-3, IMR-32 and SK-N-F1 (Figs. 1A and S1). In this group of cell lines, the mRNA and protein levels of KDM5D were examined in relation to CDDP resistance and CDDP treatment. The RT-qPCR results showed a loss of KDM5D expression in all CDDP-resistant cell lines. In addition, CDDP treatment decreased the expression of KDM5D in sensitive cells and this decrease was dependent on the duration of incubation with CDDP. The present study observed that no expression was present in drug-resistant cell lines even after treatment with CDDP (Figs. 1B and S1). The same results were observed at the protein level, as determined by means of flow cytometry (Figs. 1C and S1, S2, S3, S4, S5, S6, S7). For further experiments, UKF-NB-3 and UKF-NB-3CDDP cell lines were selected, as they were used previously to conduct a study with this cell line on the importance of KDM5B for CDDP-resistance (18).

Figure 1. Loss of KDM5D expression in CDDP-resistant neuroblastoma cell line UKF-NB-3CCDDP. PrestoBlue assay results showing cytotoxicity of CDDP after 72 h. (A) UKF-NB-3 has an IC50 of 13.33±0.97 µM and CDDP-resistant UKF-NB-3CDDP has an IC50 of 32.39±0.96 µM. (B) KDM5D mRNA expression was lost in CDDP-resistant UKF-NB-3CDDP. Treatment with 20 µM CDDP for 72 h significantly decreased KDM5D levels in UKF-NB-3. The mRNA expression was detected by reverse transcription-quantitative PCR. (C) KDM5D protein expression was detected by flow cytometry in UKF-NB-3 and CDDP-resistant UKF-NB-3CDDP cell lines and showed the same pattern of expression as mRNA levels. Data are shown as mean ± standard deviation from three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test. **P<0.01; ***P<0.001 (ANOVA with Tukey's post hoc). NB3, UKF-NB-3; NB3 CDDP, CDDP-resistant cell line UKF-NB-3CDDP.

KDM5D expression decreases histone H3K4 trimethylation in neuroblastoma cells

H3K4me3 is frequently associated with transcriptional activation of neighboring genes and therefore could activate oncogenes (64,65). We observed the change in trimethylation of H3K4 after CDDP treatment, silencing or overexpression of KDM5D in neuroblastoma cell lines (Fig. S8). To demonstrate the importance of KDM5D for methylation, the present study monitored H3K4 trimethylation after KDM5D silencing and overexpression. siRNA against KDM5D in the cell line UKF-NB-3 decreased expression levels of KDM5D mRNA (measured by RT-PqCR; Fig. 2A) and protein (measured by flow cytometry; Figs. 2B and S9) compared with non-coding siRNA-transfected control cells. Treatment of UKF-NB-3 with CDDP reduced KDM5D expression in controls, but did not further reduce expression in siRNA-transfected cells (Figs. 2C, D and S10). In addition, trimethylation of histone H3K4 was significantly increased in the KDM5D knockdown cells compared with the UKF-NB-3 control cells as shown by flow cytometry results, whereas the treatment with CDDP increased trimethylation only in the transfected cells, but not in control (Figs. 2E, F and S11).

Figure 2. KDM5D knockdown affects histone H3K4 trimethylation in CDDP-sensitive neuroblastoma cell line UKF-NB-3. (A) Reverse transcription-quantitative PCR showed decreased expression of KDM5D mRNA in UKF-NB-3 cell line transfected with siKDM5D #1 (12.5 nM) or siKDM5D #2 (25 nM) compared to control NC #1 (12.5 nM) or NC #2 (25 nM) (P<0.001). (B) Reduced expression of KDM5D protein after siKDM5D transfection (48 h) was detected by flow cytometry (P<0.001). (C) Flow cytometry histogram showed decreased expression of KDM5D protein after siKDM5D transfection (48 h) and treatment with CDDP (48 h). (D) Flow cytometry showed decreased expression of KDM5D protein in control after treatment with CDDP in UKF-NB-3 (P<0.001) but did not further decrease the expression of KDM5D in transfected cells. (E) Flow cytometry histogram showed increased expression levels of histone H3K4 trimethylation after siKDM5D transfection (48 h). (F) Silencing of KDM5D increased the level of histone H3K4me3 trimethylation (P<0.01) and treatment with CDDP in UKF-NB-3 did not increase the level of H3K4me3 in control but further increased the level of histone H3K4me3 trimethylation in transfected cells (P<0.001). Data are shown as mean ± standard deviation from three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test. *P<0.05; **P<0.01; ***P<0.001. CTR, control; si, short interfering RNA; SEC, control with only the secondary antibody; NC, control cells with non-coding RNA; siKDM5D, control cells transfected with siKDM5D; FC, flow cytometry.

The CDDP-resistant cell line UKF-NB-3CDDP, which does not express KDM5D, was transfected with the KDM5D ORF clone plasmid pCMV-3Tag1 carrying the KDM5D gene for 48 h, resulting in overexpression of KDM5D at the protein level compared with cells transfected with the control plasmid (Figs. 3A and S12). In addition, treatment with CDDP decreased the expression of KDM5D in transfected cells (Figs. 3B, C and S13). Trimethylation of histone H3K4 was significantly decreased in cells transfected with KDM5D compared with control, while treatment with CDDP increased H3K4 trimethylation in both control and transfected cells (Figs. 3D, E and S14).

Figure 3. Aberrant expression of KDM5D affects histone H3K4 trimethylation in the CDDP-resistant neuroblastoma cell line UKF-NB-3CDDP. (A) Flow cytometry showed increased levels of KDM5D protein in the UKF-NB-3CDDP cell line transfected with the KDM5D ORF-clone plasmid pCMV-3Tag1, expressing the KDM5D gene (pl-KDM5D #, 8 ng/µl; or pl-KDM5D #2, 16 ng/µl) compared with control cells transfected with the control plasmid pCMV6-AC-GFP (mock #1, 8 ng/µl; or mock #2, 16 ng/µl), while control cells did not express KDM5D (P<0.001). (B) Flow cytometry histogram showed increased expression of KDM5D protein after KDM5D overexpression (48 h). (C) Flow cytometry proved increased protein level of KDM5D in KDM5D transfected UKF-NB-3CDDP cells and showed that CDDP treatment in transfected cells decreased protein expression of KDM5D (P<0.001). (D) Flow cytometry histogram showed that histone H3K4 trimethylation decreased after KDM5D overexpression (48 h). (E) KDM5D overexpression decreased histone H3K4 trimethylation in UKF-NB-3CDDP (P<0.001) but treatment with CDDP increased H3K4me3 trimethylation in transfected cells (P<0.05). Data are shown as mean ± standard deviation from three independent experiments. Statistical significance was determined using one-way (A) and two-way ANOVA (C, E) with Tukey's post hoc test **P<0.01; ***P<0.001. CTR, control; pl-ORF-clone plasmid; SEC, control with only the secondary antibody; Mock/NC, control cells with non-coding RNA; pl-KDM5D, control cells transfected with pl-KDM5D; FC, flow cytometry.

KDM5D expression increases the cell sensitivity to CDDP

To determine the effect of KDM5D expression on sensitivity to CDDP in neuroblastoma, the percentage of viable cells following treatment with CDDP at varying concentrations and time intervals was monitored. KDM5D knockdown did not affect the viability of cells in UKF-NB-3 after 24 h of transfection (Fig. S15) or after 48 h of transfection (Fig. 4A). The viability of KDM5D knockdown UKF-NB-3 cells decreased after incubation with CDDP depending on incubation time and concentration, but the decrease in viability of NC transfected cells after treatment with CDDP was more substantial (Figs. 4A and S16).

Figure 4. KDM5D expression affects cell sensitivity to CDDP in UKF-NB-3 and UKF-NB-3CDDP. PrestoBlue assay results showed that incubation with CDDP (48 h) after (A) KDM5D silencing (48 h) in UKF-NB-3 caused reduced inhibition of cell viability induced by CDDP in silenced cells compared with NC after treatment with increasing concentrations of CDDP (P<0.001). (B) Overexpression of KDM5D in UKF-NB-3CDDP made the cells more sensitive to CDDP compared with mock (P<0.5; P<0.001). Data are shown are mean ± standard deviation from three independent experiments. Statistical significance was determined using two-way ANOVA with Tukey's post hoc test. *P<0.05; **P<0.01; ***P<0.001 (ANOVA with Tukey's post hoc test). CTR, control; si, short interfering RNA; pl, ORF-clone plasmid.

To compare the effect of inhibition of KDM5s demethylases on the viability and sensitivity of UKF-NB-3 and UKF-NB3CDDP cells to CDDP, KDOAM-25 citrate was used as an inhibitor of the KDM5 demethylases subfamily (66), and therefore a concentration that effectively reduced H3K4 trimethylation without influencing cell survival (400 nM) was selected (data not shown). Inhibition of KDM5 by KDOAM-25 protected both sensitive (UKF-NB-3) and resistant (UKF-NB-3CDDP) cells from the effects of CDDP (Fig. S16).

Overexpression of KDM5D did not alter cell viability in UKF-NB-3CDDP after 24 h, even after incubation with CDDP (Fig. S17). However, after 48 h of incubation with CDDP, cell viability decreased with increasing CDDP concentration compared with mock-transfected controls, whose viability did not change after treatment with CDDP (Fig. 4B).

The decreased sensitivity to CDDP after incubation with the inhibitor was consistent with the downregulation of KDM5D by siRNA, and conversely, the increase in sensitivity to CDDP in the resistant line with induced KDM5D expression suggests the importance of KDM5D in the anticancer efficacy of CDDP.

Expression of KDM5D increases CDDP induced apoptosis in neuroblastoma cells

To determine whether overexpression of KDM5D in resistant cells and CDDP treatment induces apoptosis, activated caspase-3 and cleavage of PARP was examined to determine the relationship between the expression of KDM5D and CUL4A. Control cells had increased levels of activated caspase-3 after treatment with CDDP. Silencing of KDM5D in the sensitive cell line UKF-NB-3 did not change the amount of activated caspase-3 compared with control cells. Treatment with CDDP increased activated caspase-3, but the level of cleaved caspase-3 was significantly lower in cells with KDM5D knockdown compared with controls after CDDP treatment (Figs. 5A, S18 and S19). The KDM5 pan-inhibitor KDOAM-25 citrate had a similar effect as KDM5D silencing (Fig. S20, Fig. S21, Fig. S22). Overexpression of KDM5D resulted in an increase in activated caspase-3 level after treatment with CDDP in contrast with control cells (Figs. 5B and S23). Taken together, KDM5D is involved in the sensitivity to CDDP induced apoptosis in neuroblastoma cells.

Figure 5. Expression of KDM5D increases CDDP induced apoptosis in neuroblastoma cells. (A) Cleaved caspase-3 in CDDP sensitive cell line UKF-NB-3 and (B) CDDP resistant cell line UKF-NB-3CDDP. Sensitive cell line UKF-NB-3 had increased levels of activated caspase-3 after CDDP treatment (P<0.001), where silencing of KDM5D led to decreased levels compared with NC (P<0.001). In CDDP-resistant cell line UKF-NB-3CDDP, CDDP treatment after transfection with plasmid with KDM5D gene, increased activated caspase-3 level (P<0.01) compared with mock. Data are shown as mean ± standard deviation from three independent experiments. Statistical significance was determined using two-way ANOVA with Tukey's post hoc test **P<0.01; ***P<0.001. si, short interfering RNA; pl, ORF-clone plasmid; Mock/NC, control cells with non-coding RNA; siKDM5D/pl-KDM5D, control cells transfected with siKDM5D/pl-KDM5D; FC, flow cytometry.

KDM5D expression decreased S-phase, cell proliferation and migration

Because inhibition of KDM5D protects cells from the effect of CDDP (Figs. 4A, 5A, S15 and S16) and artificial expression, on the contrary, increases the effect of this cytostatic agent (Figs. 4B, 5B and S17), the present study focused on the significance of KDM5D for cell cycle and proliferation. In the sensitive cells, silencing of KDM5D resulted in an increase of cells in S phase and a decrease in G2/M phase compared with the control (Figs. 6A, S24 and C). Transfection of the plasmid with KDM5D in the cell line UKF-NB-3CDDP decreased the percentage of cells in S phase and increased G0/G1 phase compared with control (Figs. 6B, S25A and E). Treatment with CDDP resulted in cell cycle arrest at checkpoint G0/G1 and G2/M in the sensitive cell line, and therefore the cell cycle was not evaluable (Fig. S24B, D, F).

Figure 6. Flow cytometry analysis of cell cycle, proliferation and cell migration by xCELLigence in CDDP-sensitive cell line UKF-NB-3 and CDDP-resistant cell line UKF-NB-3CDDP. (A) Silencing of KDM5D in sensitive cell line UKF-NB-3 led to increased S phase (P<0.01) and decreased G2/M phase (P<0.05) compared with control or cells with NC. (B) Transfection of plasmid with KDM5D in CDDP-resistant cell line UKF-NB-3CDDP led to decreased S phase (P<0.001) and increased G0/G1 phase (P<0.01) compared with control or mock (P<0.001). (C) Monitoring of cell migration by xCELLigence after transfection of siKDM5D in UKF-NB-3 or ORF cDNA plasmid with KDM5D gene in UKF-NB-3CDDP. (D) Monitoring of cell proliferation by xCELLigence after transfection of siKDM5D in UKF-NB-3 or ORF cDNA plasmid with KDM5D gene in UKF-NB-3CDDP. Data are shown as mean ± standard deviation from three independent experiments. Statistical significance was determined using one-way ANOVA (A) and two-way ANOVA (B, C and D) with Tukey's post hoc test. *P<0.05; **P<0.01; ***P<0.001 (ANOVA with Tukey's post hoc test). CTR, control; NC, non-coding RNA; si, short interfering RNA; pl, ORF-clone plasmid; NB3, UKF-NB-3; FC, flow cytometry.

The xCELLigence system was used to monitor cell migration (Figs. 6C and S26A) and proliferation (Figs. 6D and S26B-D) in real time, which showed that the CDDP-resistant UKF-NB-3CDDP had a higher cell index of migration and proliferation compared with the CDDP-sensitive UKF-NB-3 with KDM5D expression. In addition, KDM5D knock down increased cell proliferation even in IMR-32 and SK-N-F1 lines (Fig. S26C-D). The silencing of KDM5D in UKF-NB-3 increased cell migration and proliferation, while KDM5D overexpression in UKF-NB-3CDDP cells decreased migration and proliferation compared with controls. KDM5D silencing was verified over a period of 7 days using western blotting (Fig. S26E).

KDM5D plays a role in the development of chemoresistance to CDDP in neuroblastoma through regulation of the CUL4A gene

To assess the relationship between CUL4A and KDM5D (Fig. S18), its expression in CDDP-sensitive and resistant cells was examined. The CDDP-sensitive cell line UKF-NB-3 showed reduced expression of CUL4A protein compared with the CDDP-resistant UKF-NB-3CDDP, and the expression of CUL4A increased after treatment with CDDP in the sensitive but not in the resistant cells (Figs. 7A, B S27 and S28). In addition, knockdown of KDM5D increased CUL4A expression Figs. 7B and S27). In UKF-NB-3CDDP cells, overexpression of KDM5D decreased CUL4A expression compared with control, while CDDP treatment of cells with overexpressed KDM5D increased CUL4A expression levels. However, in the control, CDDP treatment did not alter CUL4A expression in resistant cells (Figs. 7C and S28). Inhibition of all KDM5s members by KDOAM-25 in sensitive cells increased level of CUL4A, compared with control but did not change CUL4A expression levels in chemoresistant cells that do not express KDM5D (Figs. S11 and S29, S30, S31).

Figure 7. KDM5D and CDDP treatment affect expression of CUL4A in UKF-NB-3 and UKF-NB-3CDDP. Flow cytometry analysis in (A) UKF-NB-3 and UKF-NB-3CDDP, where sensitive cell line UKF-NB-3 showed lower expression of CUL4A gene compared with the resistant cell line (P<0.05) and this expression increased in sensitive cells after CDDP treatment (P<0.001) compared with control. (B) Silencing of KDM5D increased of CUL4A level (P<0.001). (C) In the CDDP-resistant cell line UKF-NB-3CDDP, KDM5D overexpression decreased CUL4A expression compared with mock and control plasmid (P<0.001). CDDP treatment of overexpressed cells with KDM5D increased the level of CUL4A (P<0.001), while in control cells CDDP treatment did not change the level of CUL4A. Data are shown as mean ± standard deviation from three independent experiments. Statistical significance was determined using two-way ANOVA with Tukey's post hoc test. *P<0.05; **P<0.01; ***P<0.001 (ANOVA with Tukey's post hoc test). CTR, control; NC, non-coding RNA; si, short interfering RNA; pl, ORF-clone plasmid; NB3, UKF-NB-3; Mock/NC, control cells; siKDM5D/pl-KDM5D, control cells transfected with siKDM5D/pl-KDM5D; FC, flow cytometry.

Reduced expression of KDM5D is associated with poor overall survival

To investigate the role of KDM5D and CUL4A in neuroblastoma, patient data was screened from the R2 database: Genomics Analysis and Visualization Platform (http://r2.amc.nl). The association between KDM5D expression and overall survival was compared in male patients with two different neuroblastoma risk groups according to International Neuroblastoma Staging System (INSS) stages: non-high-risk (st1, st2, st3 and st4S) and high-risk (st4) (67). This analysis showed that low KDM5D expression was associated with poor survival rates in the group with st4 (Figs. 8B and S32). Analysis of three different datasets of patients with neuroblastoma from the R2 platform indicated a statistically significant correlation between KDM5D expression and higher survival rates in high-risk neuroblastoma patients. In the non-high-risk group, there was not a significant difference in all data sets (Figs. 8A and S32). These observations are consistent with our hypothesis that low KDM5D concentration in neuroblastoma has tumor-promoting effects and is associated with chemoresistance.

Figure 8. Kaplan-Meier curves analyzing KDM5D (A, B, C) and CUL4A (D, E, F) expression data and overall survival of patients in the SEQC neuroblastoma dataset (498; RPM; seqcnb1): (A) Male patients with stages 1, 2, 3 and 4S, KDM5AD expression. (B) Male patients with stage 4, KDM5D expression. (C) All male patients according to the International Neuroblastoma Staging System, KDM5D expression. (D) Male patients with stages 1, 2, 3 and 4S, CUL4A expression. (E) Male patients with stage 4, CUL4A expression. (F) All male patients according to the International Neuroblastoma Staging System, CUL4A expression. Statistical significance of survival differences was determined using the log-rank (Mantel-Cox) test. R2: Genomics Analysis and Visualization Platform revealed that low relative expression of KDM5D and high expression of CUL4A is associated with worse survival.

To uncover the relationship between the expression of KDM5D and CUL4A, the present study further analyzed the data from the aforementioned databases in R2: for an association between the expression of CUL4A and overall survival of neuroblastoma patients. Kaplan-Meier analysis in these datasets revealed that high relative expression of CUL4A is often associated with worse survival in two datasets of patients with stages 1, 2, 3 or 4S (Figs. 8D and S33); moreover, this association is significant in stage 4 (Figs. 8E and S32), however, this was not true in the Oberthuer dataset.

Discussion

It is well known that in cancer, aberrant epigenetic modifications, which include histone methylation, contribute to various stages of neoplastic development, including initiation, promotion, invasion, metastasis, EMT and chemoresistance (1–5). There is increasing evidence for the importance of histone lysine demethylase function, the dysregulation of which has been described in several types of cancer (8,9). Aberrant expression of KDM5D has been observed in a number of types of cancer (27–33). According to the results of analysis of publicly available genomics data, the present study found that low expression of KDM5D is associated with worse survival in male patients with stage 4 neuroblastoma. As drug resistance often develops in high-risk neuroblastoma (53,54), the present study investigated the role of KDM5D in the development of chemoresistance to CDDP, which is commonly used in therapy of the high-risk neuroblastoma (68).

The present study demonstrated that expression of KDM5D was lost in all tested CDDP-resistant neuroblastoma cell lines compared with sensitive parental cells. Silencing of KDM5D in the sensitive neuroblastoma cell lines resulted in increased trimethylation of H3K4, decreased sensitivity of cells to CDDP, inhibition of apoptosis induced by CDDP, increased proliferation and proportion of cells in S phase and acceleration of cell migration. To date, limited studies have been published describing the relationship between KDM5D and chemoresistance. Komura et al (27) found that KDM5D plays an important role as a tumor-suppressor in castration-resistant prostate cancer, where it activates the androgen receptor, and low KDM5D expression associates with docetaxel insensitivity, aggressiveness and worse prognosis. KDM5D, along with five other Y-linked genes, is reported to be downregulated in 12 major non-reproductive types of cancer, suggesting selection against their activity and their function as tumor-suppressors (69,70). Moreover, loss of the Y chromosome was described as being associated with shorter overall survival and resistance to radiotherapy and cisplatin-based chemotherapeutics in male neck squamous cell carcinoma (66,71). The present study also observed that the CDDP-resistant neuroblastoma cell line had increased expression of CUL4A compared with the sensitive parental cells and that KDM5D knockdown in the sensitive cells increased CUL4A.

The present study concludes that low expression of KDM5D is associated with worse survival in male patients with neuroblastoma, as shown by analysis of publicly available genomics data. Accordingly, the present study examined neuroblastoma cell lines with genotype XY and showed that all CDDP-resistant cell lines had KDM5D expression below the detection limit. Silencing of KDM5D results in increased trimethylation of histone H3K4 or vice versa overexpression decreased H3K4 trimethylation in CDDP-resistant cells. Our previous study demonstrated an increase in H3K4me3 after silencing of KDM5B (18), therefore all members of the KDM5 subfamily may potentially have an important function in regulating of H3K4 methylation.

Results of the present study demonstrate that KDM5D expression affects proliferation, migration and sensitivity of neuroblastoma cells to CDDP. Both the reduction of KDM5D expression in cells due to cytostatics and the more selective toxicity of cytostatics towards cells with increased expression of KDM5D and thus the selection of cells with lower expression can be applied. The concentration of CDDP (20 µM) used markedly reduced cell viability compared with controls after a 24 h incubation, and KDM5D expression values measured by RT-qPCR at this time interval were reduced insignificantly. From this, it is difficult to draw conclusions about which of the mechanisms applies.

Finally, the present study examined the expression of the gene CUL4A. Sensitive cell lines showed reduced expression of CUL4A compared with resistant ones. Moreover, this expression increased after silencing of KDM5D, CDDP or KDOAM-25 treatment in CDDP-sensitive but not in resistant cells, which do not express KDM5D. These findings suggest that CDDP and KDOAM-25 affect CUL4A expression via KDM5D. The relationship between the expression of KDM5D and CUL4A was described by Shen et al (34) in a study demonstrating that KDM5D regulates the methylation of H3K4 in the promoter of CUL4A and represses the expression of CUL4A. This study revealed that CUL4A expression plays an important role in metastasis and EMT of gastric cancer cells but to the best of our knowledge, there is no information regarding the importance of CUL4A in neuroblastoma.

Englinger et al (53) showed that loss of CUL4A expression leads to hypersensitivity to CDDP in colon cancer cells. Downregulation of CUL4A mediated a lack of nucleotide excision repair that caused trabedectin resistance and collateral CDDP hypersensitivity in colorectal carcinomas.

Overall, in CDDP-resistant neuroblastoma cell lines, there is a loss of KDM5D expression that leads to a more aggressive phenotype of neuroblastoma by promoting cell proliferation and migration, evading cell death, promoting S phase of the cell cycle and desensitizing susceptible cells to CDDP. The data of the present study may suggest that the change in expression is caused by demethylation of H3K4 in the CUL4A gene. The precise mechanisms by which KDM5D and CUL4A are involved in the development of chemoresistance remain to be elucidated. To the best of our knowledge, the relationship between KDM5D and CUL4A in neuroblastoma has not yet been studied. We hypothesize that KDM5D and/or CUL4A may be a useful biomarker for detection of chemoresistance in clinical practice and that inhibition of CUL4A may be a potential therapeutic approach in neuroblastoma after further preclinical and clinical studies.

Supplementary Material

Supporting Data

Supporting Data

Acknowledgements

Not applicable.

Funding

This research was funded by the Grant Agency of Charles University, Czech Republic (grant no. 822219) and by Ministry of Health of the Czech Republic (grant no. NW24-03-00101).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

TE conceived and led the present study. NP performed sample preparation, RT-qPCR, flow cytometry, cell proliferation assay, siRNA and ORF cDNA transfection, cell proliferation monitoring, data analysis, and the statistical analysis, and wrote the manuscript. JH contributed to the acquisition of data by performing part of the flow cytometry experiments and assisting in data interpretation. MB contributed to the acquisition of data by preparing biological samples and performing part of the RT-qPCR experiments, and assisted in data interpretation. NP, TE, JH and MB confirm the authenticity of all the raw data. All authors have reviewed the manuscript and read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References