Deubiquitinase USP7 stabilizes KDM5B and promotes tumor progression and cisplatin resistance in nasopharyngeal carcinoma through the ZBTB16/TOP2A axis

doi:10.21203/rs.3.rs-2963339/v1

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Deubiquitinase USP7 stabilizes KDM5B and promotes tumor progression and cisplatin resistance in nasopharyngeal carcinoma through the ZBTB16/TOP2A axis

https://doi.org/10.21203/rs.3.rs-2963339/v1

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published 29 Jan, 2024

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Cisplatin-based chemotherapy improves the control of distant metastases in patients with nasopharyngeal carcinoma (NPC); however, around 30% of patients fail treatment due to acquired drug resistance. Epigenetic regulation is known to contribute to cisplatin resistance; nevertheless, the underlying mechanisms remain poorly understood. Here, we showed that lysine-specific demethylase 5B (KDM5B) is overexpressed and correlates with tumor progression and cisplatin resistance in patients with NPC. We also showed that specific inhibition of KDM5B impairs the progression of NPC and reverses cisplatin resistance, both in vitro and in vivo. Moreover, we found that KDM5B inhibits the expression of ZBTB16 by directly reducing H3K4me3 at the ZBTB16 promoter, which subsequently increases the expression of Topoisomerase II- α (TOP2A) to confer cisplatin resistance in NPC. In addition, we showed that the deubiquitinase USP7 is critical for deubiquitinating and stabilizing KDM5B. More importantly, the deletion of USP7 increases sensitivity to cisplatin by disrupting the stability of KDM5B in NPC cells. Therefore, our findings demonstrate that USP7 stabilizes KDM5B and promotes cisplatin resistance through the ZBTB16/TOP2A axis, suggesting that targeting KDM5B may be a promising cisplatin-sensitization strategy in the treatment of NPC.

KDM5B

USP7

TOP2A

cisplatin resistance

nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is the most common head and neck cancer derived from nasopharyngeal epithelial cells, with a high incidence in Southern China and Southeast Asia ^{[1, 2]}. Due to the concealed position of the nasopharynx, early symptoms of NPC are difficult to detect, the majority of NPC patients are diagnosed at advanced stage ^[3]. At present, the most effective treatments for NPC include radiotherapy and/or radiotherapy combined with adjuvant chemotherapy ^[4]. Cisplatin-based simultaneous/adjuvant radiotherapy and chemotherapy is considered to be the standard treatment for locally advanced NPC ^{[5, 6]}. However, cisplatin resistance is one of the main obstacles in the treatment of locally advanced NPC ^[7], and the molecular mechanisms underlying the cisplatin sensitivity of NPC are far from fully understood.

KDM5B (lysine specific demethylase 5B), also known as PLU-1 or JARID1B, is a member of a sub-family of the JMJD family (jmjc-KDMs) that removes methyl groups from H3K4me2/3 ^[8]. By catalyzing demethylation of H3K4me2/3 to inhibit the expression of multiple genes ^[9–11], KDM5B regulates many cellular processes, including cell stemness ^[12], DNA repair ^[13–15], and cancer immunity ^{[16, 17]}. KDM5B is overexpressed, amplified or mutated in many types of cancer, and promotes tumor progression ^[18]; however, the role of KDM5B in cisplatin resistance in patients with NPC remains unclear.

Topoisomerases are enzymes responsible for overcoming topological problems in the process of DNA replication, transcription and repair, and has been identified as a target for antitumor therapy ^[19]. Topoisomerase II- α (TOP2A) plays an important role in genome structure and chromosome separation ^[20]. In many different cancers, the gene encoding TOP2A is significantly increased ^[21]. The activation of TOP2A also promotes resistance to platinum-based chemotherapy in cancer ^[22]. Therefore, a deeper understanding of the regulatory mechanisms of TOP2A may help to elucidate the mechanisms of cisplatin resistance in NPC.

In this study, we demonstrated that KDM5B promotes the progress of NPC and resistance to cisplatin in vivo and in vitro. Cisplatin resistance induced by KDM5B may be related to the upregulation of TOP2A. We also identified a novel KDM5B deubiquitinase, USP7, which can enhance the stability of KDM5B in NPC cells. We also showed that KDM5B inhibits the expression of ZBTB16, a transcriptional repressor of TOP2A, thereby regulating the expression of TOP2A. Finally, we found that the USP7/KDM5B/ZBTB16/TOP2A axis contributed to the sensitivity of NPC cells to cisplatin treatment.

Cell lines

Human NPC cell lines HNE1 and CNE2 were obtained from the Sun Yat-sen University Cancer Center (Guangzhou, China) ^[23]. Nasopharyngeal carcinoma cell lines were maintained in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; AC03L055, Shanghai Life-iLab Biotech, China) and 100 U/ml each of penicillin and streptomycin. All cells were placed in the incubator at 37°C in 5% CO2.

Antibodies and reagents

KDM5B (#A14104, ABclonal, 1:500 dilution), USP7 (#66514-1-Ig, Proteintech, 1:5000 dilution), GAPDH (#60004-1-Ig, Proteintech, 1:10000 dilution), ZBTB16 (#66672-1-Ig, Proteintech, 1:2000 dilution), TOP2A (#20233-1-AP, Proteintech, 1:10000 dilution), Flag-tag (#66008-4-Ig, Proteintech, 1:5000 dilution), H3K4me3 (#A22146, ABclonal, 1:10000 dilution), and HA-tag (#51064-2-AP, Proteintech, 1:5000 dilution) were employed for western blot analysis. KDM5B (#A14104, ABclonal, 1:500 dilution), and USP7 (#66514-1-Ig, Proteintech, 1:500 dilution) were used for the immunohistochemistry of tissue microarray (#HNasN132Su01). Cisplatin (#S1166), and MG132 (#S2619) were obtained from the Selleck (Shanghai, China). Tissue microarray slides (#HNasN132Su01) were purchased from Outdo Biotech, Shanghai, China. The method of scoring the staining intensity was described previously ^[24].

Western blot analysis and co-immunoprecipiation (co-IP)

The collected cells were lysed with RIPA buffer (#G2002, servicebio, China), containing phosphatase inhibitors (#G2007, servicebio, China) and 1% protease inhibitors (#G2007, servicebio, China). Then the cell lysates were centrifuged at 12,000 rpm at 4°C for 25 minutes and collected the supernatants. And 5 × loading buffer was added to the supernatants and boiled 10min in 100°C water. The concentration of protein in cell lysates was detected by enhanced BCA protein analysis kit (#G2026, servicebio, China). The same amount of protein was loaded in each well of SDS-PAGE gels. The protein in the gels were transferred to the PVDF membranes. Then PVDF membranes were blocked with 5% non-fat milk at room temperature for 1 h and incubated with the primary antibody at 4 ℃. The next day, the membranes were washed with 1 × TBST 3 times (10 minutes each time) and incubated with the second antibody for 1 h. Then the membranes were washed again and exposed in dark field using ECL detection reagents (#G2020, servicebio, China). For co-immunoprecipiation (IP), collected cells were added with 1mL RIPA protein lysate containing 1% protease inhibitors on ice for at least 30 min. The supernatant was collected and co-cultured with Protein A and G Agarose beads (#G1718, Santa Cruz, America) and primary antibodies or IgG for 24 h at 4°C. Then, the beads were washed with NETN buffer 6 times, added with 50 uL sample loading buffer, and boiled for 15 mins. Then, these samples were subjected to western blotting analysis. The antibodies used as follows: KDM5B (#A14104, ABclonal, 1:200 dilution), USP7 (#66514-1-Ig, Proteintech, 1:500 dilution).

Plasmids and siRNA transfection and shRNA infection

USP7, KDM5B, ZBTB16, and TOP2A were obtained from WZ Bioscience (Shandong, China) and GeneChem (Shanghai, China). Flag-USP7 was cloned into the CMV-MCS-3xFlag-SV40-neomycin vector by GENECHEM (Shanghai, China). Myc-KDM5B was cloned into the pEnter vector by GENECHEM (Shanghai, China). His-ZBTB16 was cloned into the pEnter vector with C-terminal Flag and His tag by WZ Bioscience (Shandong, China). HA-TOP2A was cloned into the pCMV-N-HA vector. pGEX-4T-1 vector was used to construct GST-tag plasmids. Short hairpin RNAs (shRNAs) were obtained from GeneChem (Shanghai, China), and the siRNA was purchased from RiboBio (Guangzhou, China). siRNA transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific, China), and 50 nM siRNAs were used. At least 48 hours later, cells were harvested and analyzed. The method of shRNA transfection was described previously ^[24]. The sequences of the shRNAs and siRNAs are provided in Supplementary Table S1.

Quantitative real-time PCR (RT-qPCR) assay

In brief, RNA was extracted by using TRIzol reagent (#R401-01 RNA isolater Total RNA Extraction Reagent, vazyme, Nanjing, China). RT-qPCR was performed by using a reverse transcription kit and PCR kit (#R323-01 HiScript III RT SuperMix for qPCR, #Q111-02 AceQ qPCR SYBR Green Master Mix, vazyme, Nanjing, China) referring to the the manufacturer’s instructions. GAPDH served as the reference gene. The primer sequences for RT-qPCR are provided in Supplementary Tables S2.

Chromatin immunoprecipitation (ChIP) and ChIP-qRT-PCR

For ChIP-qPCR, ChIP Assay Kit (#P2078, Beyotime, China) were used, following the protocol of the manufacturer’s instructions. Immunoprecipitation was performed using the appropriate antibodies as follows: KDM5B (#15327, Cell Signaling Technology, 1:50 dilution), H3K4me3 (#9751, Cell Signaling Technology, 1:50 dilution), and ZBTB16 (#66672-1-Ig, Proteintech, 1:50 dilution) were used for ChIP assay. Mouse IgG antibody (#61656S, Cell Signaling Technology, 1:1000 dilution) and rabbit IgG antibody (#3900S, Cell Signaling Technology, 1:1000 dilution) were used as a negative control. The primers were designed according to the promoter sequences of the genes of interest. The sequences of the ChIP-qPCR primers are shown in Supplementary Table S3.

In vitro cell proliferation assay and CCK-8 assay

Cell proliferation assay was performed following the manufacturer’s instruction of the Cell Counting Kit-8 (CCK-8). Briefly, 1 × 103 cells were seeded in 96-well plates and cultured with 100 µL medium. 20 µL of the CCK-8 reagent (#C0037, Beyotime) was added to each well one hour before the end of the incubation period following the manufacturer’s instructions. The absorbance values at 450 nm were measured using a Multimode Plate Reader (EnSpire® 2300, USA). CCK-8 assay was applied to measure the half maximal inhibitory concentration (IC₅₀) of cisplatin after treated with a serial dose of cisplatin for 24 h in HNE1 and CNE2 cells.

Cell invasion assay

Transwell chambers (8.0 µm Pore Size, CORNING) were used to assess the cell invasion ability. Cells were seeded into the Matrigel-coated invasion upper chamber with serum-free 1640 medium, and Medium containing 30% FBS was placed in the lower chambers and incubated for 18 hours in cell incubator. After incubation, the cells were fixed in methanol for 30 mins and then stained with Crystal Violet stain solution (#C0121, Beyotime). Invasion cells were evaluated under a microscope at five fields per well. All the experiments were repeated three times.

Colony formation assay

In this study, cell proliferation was evaluated using the colony formation assay. Cells were seeded into 6-well plates (1000 cells per well) and cultured in the 1640 medium containing 10% FBS at 37°C. After 2 weeks, the cells were fixed in methanol for 30 min, stained with Crystal Violet Staining Solution for 20 mins, and washed 3 times with 1 × PBS. The number of colonies was counted and recorded. All the experiments were repeated three times.

Transcriptome sequencing (RNA-seq)

Total RNA was extracted by using TRIzol reagent (#R401-01 RNA isolater Total RNA Extraction Reagent, vazyme, Nanjing, China), and transcriptome sequencing was performed by NOVOGENE (Beijing, China) based on the Illumina platform. The library preparations were sequenced on an Illumina NovaSeq platform, and 150 bp paired-end reads were generated. The RNA-seq data is provided in Supplementary Tables S4.

In vivo tumor growth study

Ethical approval was obtained by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology for all animal procedures. BALB/c nude mice (4–5 weeks old) were purchased from Shulaibao Biotech (Wuhan, China). Mice were randomly divided into groups (at least n = 5). HNE1 cells (5 × 10⁶) treated in different manners were collected and inoculated subcutaneously into the left dorsal flank of nude mice. Tumor volume was calculated using the formula (L × W²)/2.

Statistical analysis

Each assay was performed in at least three independent experiments. Data were presented as the mean ± SD. GraphPad Prism 8 software was used to calculate the P value using Student’s t-test for between-group comparisons and one-way ANOVA or two-way ANOVA for multiple comparisons. P value < 0.05 was considered statistically significant. In all cases, the significance of differences was indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; not significant (ns), P > 0.05.

Abnormal KDM5B expression promotes proliferation, invasion and cisplatin resistance in NPC cells.

In order to identify key genes responsible for regulating histone methylation status in NPC, we conducted a bioinformatics analysis utilizing public databases. In total, we identified 365 genes involved in histone methylation-related biological processes and signaling pathways in the Molecular Signatures Database (MSigDB). Next, we combined the transcriptome data of the GSE118719 and GSE53819 datasets and identified 5 histone methyl modifier genes, of which KDM5B ranked the highest (all P < 0.05; Fig. 1a, b). Further, KDM5B was significantly upregulated in tumor tissues compared with tumor-adjacent tissues in the GSE118719 and GSE53819 datasets (all P < 0.001, Fig. 1c, d). To investigate the role of KDM5B in NPC, we silenced KDM5B expression in HNE1 and CNE2 cells (Supplementary Fig. 1a, b). CCK-8, colony-formation and Transwell assays showed that KDM5B depletion suppressed NPC cell proliferation, invasion and migration in vitro (Supplementary Fig. 1c-e). Moreover, we showed that rescue of KDM5B expression in cells with KDM5B silencing reversed the suppressive effects on cell proliferation that induced by KDM5B knockdown in vitro and in vitro (Fig. 1f, g and Supplementary Fig. 1f, g).

Next, we aimed to investigate whether KDM5B contributed to cisplatin resistance in NPC cells. We showed that KDM5B silencing sensitized NPC cells to cisplatin, while KDM5B overexpression in NPC cells increased the half maximal inhibitory concentration (IC₅₀) for cisplatin (Fig. 1k, l). Moreover, CCK-8 and colony-formation assays showed that KDM5B silencing enhanced the inhibitory effect of cisplatin on NPC cell proliferation (Fig. 1j and Supplementary Fig. 1h). We also performed Annexin-V/propidium iodide (PI) assay to show that KDM5B knockdown plus cisplatin treatment significantly increased apoptosis (Fig. 1h, i). Additionally, the inhibitory effect of cisplatin on NPC tumor growth was significantly increased following KDM5B knockdown (Fig. 1m). These results suggested that aberrant KDM5B expression promotes cell proliferation and cisplatin resistance in NPC.

KDM5B negatively regulates ZBTB16 expression in NPC.

To further explore the underlying mechanism by which KDM5B regulates the progression of NPC cells, we utilized small interfering RNA (siRNA) to inhibit the expression of KDM5B in HNE1 cells (Fig. 2a, b). ChIP-Atlas (http://chip-atlas.org/) was used to predict the potential downstream target genes of KDM5B. Next, we combined our RNA-seq datasets (upregulated genes) with the GSE118719 (downregulated genes) and GSE53819 (downregulated genes) databases to screen 61 candidate genes (Fig. 2c), among which ZBTB16 was the most significantly upregulated gene after KDM5B silencing (Fig. 2d). Consistently, in the GSE53819 dataset, the expression of ZBTB16 in tumor tissue was significantly downregulated compared with normal tissue (Fig. 2e), and was negatively correlated with KDM5B expression (Fig. 2f). We next assessed whether KDM5B inhibits ZBTB16 expression directly through histone demethylation in the ZBTB16 promoter region. We found that KDM5B knockdown increased the protein and mRNA levels of ZBTB16 in HNE1 and CNE2 cells (Fig. 2g). Conversely, KDM5B overexpression reduced ZBTB16 mRNA and protein levels in HNE1 and CNE2 cells (Fig. 2h). ChIP-Atlas analysis revealed a KDM5B binding peak in the promoter of ZBTB16 (Fig. 2i). We also showed that KDM5B represses the transcription of target genes by removing methyl groups from H3K4me2/3 ^[8], and H3K4me3-binding sites overlap with the KDM5B-binding sites in the promoter region of ZBTB16 (Fig. 2j). ChIP-qPCR analysis revealed the presence of KDM5B and H3K4me3 in the promoter of ZBTB16 (Fig. 2k, l), and demonstrated that KDM5B knockdown enhanced H3K4me3 binding (Fig. 2m). These results indicated that KDM5B regulates ZBTB16 expression by removing methylation from the ZBTB16 promoter.

KDM5B promotes progression and cisplatin resistance in NPC cells by directly inhibiting the expression of ZBTB16.

To explore the function of ZBTB16 in NPC, we silenced ZBTB16 expression in HNE1 and CNE2 cells (Supplementary Fig. 2a, b). CCK-8, colony-formation and Transwell assays showed that ZBTB16 depletion promoted NPC cell proliferation, invasion and migration in vitro (Supplementary Fig. 2c-e). We also showed that overexpression of KDM5B in ZBTB16 ablation cells further reduced the expression of ZBTB16 compared with knockdown of ZBTB16 alone in HNE1 and CNE2 cells (Fig. 3a, b). Co-knockdown of KDM5B and ZBTB16 attenuated both the downregulation effect of ZBTB16 induced by ZBTB16 silencing alone (Fig. 3c, d), and the promotion effect of NPC cells in vitro and in vivo induced by ZBTB16 silencing alone (Fig. 3e-h). These data suggested that KDM5B downregulates ZBTB16 to promote tumor growth in NPC. Furthermore, ZBTB16 silencing in NPC cells increased the half maximal inhibitory concentration (IC₅₀) values for cisplatin (Supplementary Fig. 2f), while ZBTB16 overexpression rendered NPC cells more sensitive to cisplatin treatment (Supplementary Fig. 2g). CCK-8 assays showed that ZBTB16 silencing decreased the inhibitory effect off cisplatin on NPC cell proliferation (Fig. 3i). Through Annexin-V/PI analysis, we showed that ZBTB16 knockdown plus cisplatin treatment significantly reduced the rate of apoptosis compared with cisplatin alone (Supplementary Fig. 2h). Notably, IC₅₀ cytotoxicity assay (Fig. 3j), colony-formation (Fig. 3k) and CCK-8 (Supplementary Fig. 2i) assay all showed that ZBTB16 silencing alone promotes cisplatin resistance in NPC cells, but this effect was weakened by co-knockdown of KDM5B and ZBTB16. These results suggested that KDM5B could promote cisplatin resistance in NPC by inhibiting the expression of ZBTB16.

ZBTB16 negatively regulates TOP2A expression in NPC.

According to previous reports, as a transcriptional regulator, ZBTB16 is unlikely to directly regulate cisplatin resistance in NPC. Therefore, we explored downstream effector molecules that might mediate cisplatin resistance. We used ChIP-Atlas to predict the potential downstream target genes of ZBTB16 and combined our RNA-seq datasets (downregulated genes) with the GSE118719 (upregulated genes) and GSE53819 (upregulated genes) databases to screen 23 candidate genes (Fig. 4a), among which TOP2A was the most significantly downregulated after KDM5B silencing (Fig. 4b). We used a volcanic map to depict the regulatory relationship between ZBTB16, TOP2A and KDM5B after KDM5B silencing (Fig. 4c). The GSE53819 dataset showed that the expression of TOP2A in tumor tissue was significantly upregulated compared with normal tissue (Fig. 4d), and was negatively correlated with KDM5B (Fig. 4e) and ZBTB16 (Fig. 4f).

It has been reported that ZBTB16 acts as a transcriptional repressor ^{[25, 26]}, so we assessed whether ZBTB16 directly inhibits the transcriptional process of TOP2A. Our results showed that ZBTB16 knockdown increased the protein and mRNA levels of TOP2A in HNE1 and CNE2 cells (Fig. 4g). Conversely, ZBTB16 overexpression reduced TOP2A mRNA and protein levels in HNE1 and CNE2 cells (Fig. 4h). Analyses using ChIP-Atlas revealed a ZBTB16 binding peak in the promoter of TOP2A (Fig. 4i, j). Subsequent ChIP-qPCR analysis indicated that ZBTB16 bound to the promoter region of TOP2A (Fig. 4k) and that knockdown of ZBTB16 attenuated this binding (Fig. 4l, n, and o) but knockdown of KDM5B enhanced this binding (Fig. 4m-o).

ZBTB16 inhibits progression and cisplatin resistance in NPC cells by directly inhibiting the transcriptional process of TOP2A.

To explore the function of TOP2A in NPC, we silenced TOP2A in HNE1 and CNE2 cells (Supplementary Fig. 3a, b). CCK-8, colony-formation and Transwell assays showed that TOP2A depletion inhibited NPC cell proliferation, invasion and migration in vitro (Supplementary Fig. 3c-e). We also showed that co-knockdown of ZBTB16 and TOP2A attenuated the downregulation effect of TOP2A induced by TOP2A silencing alone (Fig. 5a, b), and the inhibitory effect of NPC cells in vitro and in vivo induced by TOP2A silencing alone (Fig. 5c-f). Moreover, co-knockdown of ZBTB16 and KDM5B reduced the upregulation effect of TOP2A by ZBTB16 silencing alone (Fig. 5g, h). These data suggested that ZBTB16 is associated with downregulation of TOP2A and subsequent inhibition of tumor growth in NPC. Furthermore, TOP2A silencing in NPC cells reduced the IC₅₀ values for cisplatin (Supplementary Fig. 3f), while TOP2A overexpression made NPC cells more resistant to cisplatin (Supplementary Fig. 3g), and CCK-8 assays showed that TOP2A silencing enhanced the effect of cisplatin on inhibition of NPC cell proliferation (Fig. 5i). Through Annexin-V/PI analysis, we also showed that TOP2A knockdown plus cisplatin treatment significantly increased the rate of apoptosis compared with cisplatin alone (Supplementary Fig. 3h). Importantly, IC₅₀ cytotoxicity assay (Fig. 5j), colony-formation (Fig. 5k) and CCK-8 (Supplementary Fig. 3i) assays all showed that TOP2A silencing alone promoted the sensitivity of NPC cells to cisplatin treatment, but this effect was weakened by co-knockdown of ZBTB16 and TOP2A.These results suggested that ZBTB16 could enhance cisplatin sensitivity in NPC by inhibiting the expression of TOP2A.

USP7 promoted the stability of the KDM5B protein in NPC by deubiquitinating KDM5B.

We next analyzed the regulation of KDM5B in NPC. UbiBrowser (http://ubibrowser.bio-it.cn/) showed that the deubiquitinases (DUBs) USP11 and USP7 were closely associated with KDM5B (Fig. 6a). Coincidentally, we analyzed the amino acid sequence of KDM5B and found that there was a unique overlap between the USP7 binding motif and the conservative region of KDM5B (Fig. 6b). A coimmunoprecipitation (co-IP) assay showed that endogenously expressed KDM5B interacted with USP7 in both HNE1 and CNE2 cells (Fig. 6c). Consistent with this result, a GST pulldown assay demonstrated that there was an interaction between KDM5B and USP7 in vitro (Fig. 6d). We also found that KDM5B protein expression decreased or increased after knockdown or overexpression of USP7, respectively, while KDM5B mRNA remained constant (Fig. 6e, f). Furthermore, the changes in KDM5B observed after treatment with the USP7 inhibitor P5091 were consistent without observations following USP7 knockdown (Fig. 6g). We also found that USP7 knockdown using shUSP7 or P5091 could reduce the KDM5B protein expression, which was inhibited by the proteasome inhibitor MG132 (Fig. 6h, i). Moreover, the protein half-life of KDM5B was significantly decreased when USP7 was knocked down or inhibited, while overexpression of USP7 showed the opposite effect (Fig. 6j-l). We found that knocking down or inhibiting USP7 increased the polyubiquitination of KDM5B, and that overexpressing USP7 decreased the polyubiquitination of KDM5B in NPC cells (Fig. 6m, n). To analyze the protein expression of KDM5B and USP7 in tissue, we performed an NPC tissue microarray (n = 35) (Fig. 6o). The results showed a significant positive correlation between KDM5B and USP7 protein expression in samples from patients with NPC (Fig. 6p). These data suggest that USP7 promoted the stability of the KDM5B protein in NPC by deubiquitinating KDM5B.

The USP7/KDM5B/ZBTB16/TOP2A axis modulates cisplatin resistance in NPC cells.

After USP7 was inhibited or overexpressed, the expression of TOP2A also decreased or increased, respectively (Fig. 7a-c). This raised the question of whether USP7 regulated cisplatin resistance in NPC through the KDM5B/ZBTB16/TOP2A axis. To verify this hypothesis, we knocked down KDM5B expression while suppressing the expression of USP7. The results showed that TOP2A protein expression decreased, which was also observed when KDM5B alone was silenced (Fig. 7a, b). Similarly, overexpression of USP7 after KDM5B knockdown also resulted in a slight increase in TOP2A protein expression (Fig. 7c). Notably, IC₅₀ cytotoxicity assays (Fig. 7d) and colony-formation assays (Supplementary Fig. 4a, b) all showed that TOP2A silencing alone increased the sensitivity of NPC cells to cisplatin treatment, but this effect was enhanced by co-knockdown of USP7 and TOP2A. Consistently, in vitro (Fig. 7e, f and Supplementary Fig. 4c-e) and in vivo (Fig. 7g, h) assays showed that USP7 inhibitor P5091 enhanced the antitumor effect of cisplatin in NPC, and this process was enhanced after knockdown of TOP2A. In conclusion, USP7 stabilized KDM5B to inhibit the expression of ZBTB16 by demethylating H3H4 of histone, which subsequently increased the expression of TOP2A and promoted NPC cell proliferation and resistance to cisplatin (Fig. 7i).

Epigenetics plays an important role in tumor biology. In the process of tumorigenesis and progression, DNA methylation and histone post-translational modification regulate complex gene expression networks, which affect tumor growth, metastasis and drug response ^{[27, 28]}. Epigenetics has been identified as an important mechanism of tumor drug resistance ^[29]. Mechanically, epigenetic regulation plays an important role in the regulation of chromatin structure and gene expression. In particular, chromatin modification, which is responsible for chromatin remodeling and specific gene transcription, is strictly controlled by different types of epigenetic modifiers through histone post-translational modifications, including acetylation, methylation and ubiquitin ^[30].

Previous studies have shown that KDM5B can impact chromatin stability and promote drug resistance through chromatin remodeling ^{[31, 32]}. However, prior to our study, it was unknown whether KDM5B could affect the sensitivity of NPC to cisplatin through transcriptional regulation. Our bioinformatics analysis showed that KDM5B can significantly upregulate the expression of TOP2A. We also showed that KDM5B upregulates the expression of TOP2A by inhibiting the expression of ZBTB16 through demethylation of the transcriptional initiation histone H3K4, and that ZBTB16, as a transcriptional repressor, is directly involved in the transcriptional inhibition of TOP2A. As such, our data suggests that KDM5B may regulate the sensitivity of NPC to cisplatin in a TOP2A-dependent manner in vivo and in vitro.

TOP2A leads to the release of the DNA superhelix by catalyzing the transient breaking and rejoining of two strands of duplex DNA, thus altering the topology of DNA ^[33–36]. In cancer treatment, TOP2A is the main target of adriamycin, etoposide and other chemotherapeutic drugs ^{[37, 38]}. Anthracyclines, for example, can be embedded in the site where TOP2A binds to DNA, killing tumor cells by preventing broken DNA from reconnecting. Interestingly, it has been reported that TOP2A reduces DNA damage caused by the binding of platinum drugs to DNA, which can lead to drug resistance by activating nucleotide excision repair ^[39]. This suggests that TOP2A is a key mediator of cisplatin resistance in NPC cells. Importantly, the complex regulatory mechanisms underlying the activity of TOP2A require further study.

Next, we showed that USP7 bound to KDM5B to stabilize KDM5B and promoted both the progression of NPC and the development of cisplatin resistance, which were both dependent on TOP2A. We further showed that the USP7 inhibitor, P5091, enhanced the sensitivity of NPC cells to cisplatin by inducing KDM5B degradation, which provides a potential therapeutic strategy for inhibiting the KDM5B/ZBTB16/TOP2A axis in NPC. In summary, we demonstrated that upregulation of KDM5B was responsible for enhancing growth, invasion and chemoresistance in NPC cells treated with cisplatin. We also found that KDM5B, as a histone H3K4 methylation scavenger, indirectly upregulated the expression of TOP2A by inhibiting the expression of ZBTB16. Furthermore, we also demonstrated that USP7 interacted with KDM5B and prevented KDM5B from degradation in NPC cells. Thus, USP7 inhibition combined with cisplatin may have more powerful antitumor effects than treatment with cisplatin alone in patients with NPC.

Competing Interests: The authors have declared that no competing interest exists.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (82073354 to KY, 82073321 to YL, 82102201 to CW and 82102931 to YS).

Conflict of Interest

The authors have declared that no competing interest exists.

Availability of Data and Materials

The RNA-seq data is provided in Supplementary Tables S4. The datasets used and/or analyzed during the current study are available from the corresponding authors ([email protected]) on reasonable request.

Ethical Approval and Consent to participate

The study was conducted in accordance with the principles of the Declaration of Helsinki principles. It was approved by the Animal Use and Care Committees at Tongji Medical College, Huazhong University of Science and Technology.

Author Contributions

BZ designed and conceived the study; BZ, KY, YL, YS supervised the study; BZ, JL, YW performed experiments and analyzed data; JL, YW, XL, XY, ZL, SD, YD, ZZ, YT, WW, JM, YH, CW, ZZ, FH, LW, and BW provided advice and technical assistance; and SX wrote the manuscript. All authors have contributed to and approved the final manuscript.

Lin, D.-C., Meng, X., Hazawa, M., Nagata, Y., Varela, A.M., Xu, L., et al. The genomic landscape of nasopharyngeal carcinoma. Nat Genet, 46(8): 866-871. (2014)
Wei, W.I., & Sham, J.S.T. Nasopharyngeal carcinoma. Lancet, 365(9476): 2041-2054. (2005)
Chen, Y.-P., Chan, A.T.C., Le, Q.-T., Blanchard, P., Sun, Y., & Ma, J. Nasopharyngeal carcinoma. Lancet, 394(10192): 64-80. (2019)
Siegel, R.L., Miller, K.D., & Jemal, A. Cancer statistics, 2019. CA Cancer J Clin, 69(1). (2019)
Colevas, A.D., Yom, S.S., Pfister, D.G., Spencer, S., Adelstein, D., Adkins, D., et al. NCCN Guidelines Insights: Head and Neck Cancers, Version 1.2018. J Natl Compr Canc Netw, 16(5): 479-490. (2018)
Ribassin-Majed, L., Marguet, S., Lee, A.W.M., Ng, W.T., Ma, J., Chan, A.T.C., et al. What Is the Best Treatment of Locally Advanced Nasopharyngeal Carcinoma? An Individual Patient Data Network Meta-Analysis. J Clin Oncol, 35(5): 498-505. (2017)
Zhou, C., Shen, G., Yang, F., Duan, J., Wu, Z., Yang, M., et al. Loss of AKR1C1 is a good prognostic factor in advanced NPC cases and increases chemosensitivity to cisplatin in NPC cells. J Cell Mol Med, 24(11): 6438-6447. (2020)
Kooistra, S.M., & Helin, K. Molecular mechanisms and potential functions of histone demethylases. Nat Rev Mol Cell Biol, 13(5): 297-311. (2012)
Bernstein, B.E., Mikkelsen, T.S., Xie, X., Kamal, M., Huebert, D.J., Cuff, J., et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell, 125(2): 315-326. (2006)
Barski, A., Cuddapah, S., Cui, K., Roh, T.-Y., Schones, D.E., Wang, Z., et al. High-resolution profiling of histone methylations in the human genome. Cell, 129(4): 823-837. (2007)
Mikkelsen, T.S., Ku, M., Jaffe, D.B., Issac, B., Lieberman, E., Giannoukos, G., et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature, 448(7153): 553-560. (2007)
Xie, L., Pelz, C., Wang, W., Bashar, A., Varlamova, O., Shadle, S., et al. KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription. EMBO J, 30(8): 1473-1484. (2011)
Mocavini, I., Pippa, S., Licursi, V., Paci, P., Trisciuoglio, D., Mannironi, C., et al. JARID1B expression and its function in DNA damage repair are tightly regulated by miRNAs in breast cancer. Cancer Sci, 110(4): 1232-1243. (2019)
Khoury-Haddad, H., Nadar-Ponniah, P.T., Awwad, S., & Ayoub, N. The emerging role of lysine demethylases in DNA damage response: dissecting the recruitment mode of KDM4D/JMJD2D to DNA damage sites. Cell Cycle, 14(7): 950-958. (2015)
Hendriks, I.A., Treffers, L.W., Verlaan-de Vries, M., Olsen, J.V., & Vertegaal, A.C.O. SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage. Cell Rep, 10(10): 1778-1791. (2015)
Rao, M., Chinnasamy, N., Hong, J.A., Zhang, Y., Zhang, M., Xi, S., et al. Inhibition of histone lysine methylation enhances cancer-testis antigen expression in lung cancer cells: implications for adoptive immunotherapy of cancer. Cancer Res, 71(12): 4192-4204. (2011)
Zhang, S.-M., Cai, W.L., Liu, X., Thakral, D., Luo, J., Chan, L.H., et al. KDM5B promotes immune evasion by recruiting SETDB1 to silence retroelements. Nature, 598(7882): 682-687. (2021)
Xhabija, B., & Kidder, B.L. KDM5B is a master regulator of the H3K4-methylome in stem cells, development and cancer. Semin Cancer Biol, 57: 79-85. (2019)
Pilati, P., Nitti, D., & Mocellin, S. Cancer resistance to type II topoisomerase inhibitors. Curr Med Chem, 19(23): 3900-3906. (2012)
Lee, J.H., & Berger, J.M. Cell Cycle-Dependent Control and Roles of DNA Topoisomerase II. Genes (Basel), 10(11). (2019)
Chen, T., Sun, Y., Ji, P., Kopetz, S., & Zhang, W. Topoisomerase IIα in chromosome instability and personalized cancer therapy. Oncogene, 34(31): 4019-4031. (2015)
Zhu, C., Zhang, L., Zhao, S., Dai, W., Xu, Y., Zhang, Y., et al. UPF1 promotes chemoresistance to oxaliplatin through regulation of TOP2A activity and maintenance of stemness in colorectal cancer. Cell Death Dis, 12(6): 519. (2021)
Xu, S., Li, Y., Lu, Y., Huang, J., Ren, J., Zhang, S., et al. LZTS2 inhibits PI3K/AKT activation and radioresistance in nasopharyngeal carcinoma by interacting with p85. Cancer Lett, 420: 38-48. (2018)
Zhang, B., Cheng, X., Zhan, S., Jin, X., & Liu, T. MIB1 upregulates IQGAP1 and promotes pancreatic cancer progression by inducing ST7 degradation. Mol Oncol, 15(11): 3062-3075. (2021)
Melnick, A.M., Westendorf, J.J., Polinger, A., Carlile, G.W., Arai, S., Ball, H.J., et al. The ETO protein disrupted in t(8;21)-associated acute myeloid leukemia is a corepressor for the promyelocytic leukemia zinc finger protein. Mol Cell Biol, 20(6): 2075-2086. (2000)
Lin, D.-Y., Huang, C.-C., Hsieh, Y.-T., Lin, H.-C., Pao, P.-C., Tsou, J.-H., et al. Analysis of the interaction between Zinc finger protein 179 (Znf179) and promyelocytic leukemia zinc finger (Plzf). J Biomed Sci, 20(1): 98. (2013)
Carter, C.A., Zeman, K., Day, R.M., Richard, P., Oronsky, A., Oronsky, N., et al. Addressing the elephant in the room, therapeutic resistance in non-small cell lung cancer, with epigenetic therapies. Oncotarget, 7(26): 40781-40791. (2016)
Stahl, M., Kohrman, N., Gore, S.D., Kim, T.K., Zeidan, A.M., & Prebet, T. Epigenetics in Cancer: A Hematological Perspective. PLoS Genet, 12(10): e1006193. (2016)
Byler, S., & Sarkar, S. Do epigenetic drug treatments hold the key to killing cancer progenitor cells? Epigenomics, 6(2): 161-165. (2014)
Zhang, Q., & Cao, X. Epigenetic regulation of the innate immune response to infection. Nat Rev Immunol, 19(7): 417-432. (2019)
Xu, W., Zhou, B., Zhao, X., Zhu, L., Xu, J., Jiang, Z., et al. KDM5B demethylates H3K4 to recruit XRCC1 and promote chemoresistance. Int J Biol Sci, 14(9): 1122-1132. (2018)
Leadem, B.R., Kagiampakis, I., Wilson, C., Cheung, T.K., Arnott, D., Trojer, P., et al. A KDM5 Inhibitor Increases Global H3K4 Trimethylation Occupancy and Enhances the Biological Efficacy of 5-Aza-2'-Deoxycytidine. Cancer Res, 78(5): 1127-1139. (2018)
Wyles, J.P., Wu, Z., Mirski, S.E.L., & Cole, S.P.C. Nuclear interactions of topoisomerase II alpha and beta with phospholipid scramblase 1. Nucleic Acids Res, 35(12): 4076-4085. (2007)
Williamson, E.A., Rasila, K.K., Corwin, L.K., Wray, J., Beck, B.D., Severns, V., et al. The SET and transposase domain protein Metnase enhances chromosome decatenation: regulation by automethylation. Nucleic Acids Res, 36(18): 5822-5831. (2008)
Ramamoorthy, M., Tadokoro, T., Rybanska, I., Ghosh, A.K., Wersto, R., May, A., et al. RECQL5 cooperates with Topoisomerase II alpha in DNA decatenation and cell cycle progression. Nucleic Acids Res, 40(4): 1621-1635. (2012)
Lee, S., Jung, S.-R., Heo, K., Byl, J.A.W., Deweese, J.E., Osheroff, N., et al. DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg2+-mediated dynamic bending of gate-DNA. Proc Natl Acad Sci U S A, 109(8): 2925-2930. (2012)
Lu, W., Li, N., & Liao, F. Identification of Key Genes and Pathways in Pancreatic Cancer Gene Expression Profile by Integrative Analysis. Genes (Basel), 10(8). (2019)
Liu, T., Zhang, H., Yi, S., Gu, L., & Zhou, M. Mutual regulation of MDM4 and TOP2A in cancer cell proliferation. Mol Oncol, 13(5): 1047-1058. (2019)
Tsai-Pflugfelder, M., Liu, L.F., Liu, A.A., Tewey, K.M., Whang-Peng, J., Knutsen, T., et al. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22. Proc Natl Acad Sci U S A, 85(19): 7177-7181. (1988)

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Deubiquitinase USP7 stabilizes KDM5B and promotes tumor progression and cisplatin resistance in nasopharyngeal carcinoma through the ZBTB16/TOP2A axis

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Introduction

Material and Methods

Cell lines

Antibodies and reagents

Western blot analysis and co-immunoprecipiation (co-IP)

Plasmids and siRNA transfection and shRNA infection

Quantitative real-time PCR (RT-qPCR) assay

Chromatin immunoprecipitation (ChIP) and ChIP-qRT-PCR

Cell invasion assay

Colony formation assay

Transcriptome sequencing (RNA-seq)

Statistical analysis

Results

Discussion

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