ZNF582 hypermethylation promotes metastasis of nasopharyngeal carcinoma by regulating the transcription of adhesion molecules Nectin-3 and NRXN3

2.1 Clinical specimens and cell culture

In the present study, 27 normal nasopharynx tissue samples and 33 freshly frozen NPC biopsy tissue samples were obtained from the Sun Yat-sen University Cancer Center (Guangzhou, Guangdong, China). The present study was approved by the Institutional Ethical Review Board of Sun Yat-sen University Cancer Center, and informed consent was signed by all patients.

CNE2, SUNE1, HONE1, HNE1, 5-8F, and 6-10B NPC cell lines were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, ExCell Bio, Shanghai, China), and two human normal nasopharyngeal epithelial (NPE) cell lines (NP69 and N2-Tert) were maintained in keratinocyte serum-free medium (KSFM, Invitrogen) supplemented with bovine pituitary extract (BD Biosciences, Becton, NJ, USA). NP69, N2-Tert, and all NPC cell lines were generously provided by professor Mu-sheng Zeng (Sun Yat-sen University Cancer Center). HEK293T cells were grown in DMEM supplemented with 10% FBS and were obtained from American type culture collection (ATCC). All the cells were authenticated using shot-tandem repeat profiling, tested for mycoplasma contamination, and cultured for less than 2 months.

2.2 Constructs, antibodies, and reagents

ZNF582 or Nectin-3 were cloned into a Phage-puro-6tag vector via standard molecular methods. The Phage-puro-6tag vector was gifted by Dr. Xiaodong Zhang (Wuhan University, Wuhan, Hubei, China) ZNF582, Nectin-3, or NRXN3 shRNAs were cloned into the pLKO.1 vector (TranSheepBio, Shanghai, China). The Nectin-3 or NRXN3 promoter (the upstream 1,000 bp starting from the transcription starting site of Nectin-3 or NRXN3) was cloned into the pGL3-basic vector (Promega, Madison, WI, USA). The shRNA sequences are listed in Supplementary Table S1. Site-directed mutagenesis was performed with bridge-PCR and sequenced for confirmation. The anti-ZNF582 (10263, Sigma, St. Louis, MO, USA), anti-FLAG (F3165, Sigma), horseradish peroxide(HRP)-conjugated goat anti-mouse IgG (7076, Cell Signaling Technology, Boston, MA, USA), HRP-conjugated goat anti-rabbit IgG (7074, Cell Signaling Technology), anti-NRXN3 (DF9682, Affinity, Shanghai, China), anti-α-tubulin (11224-1-AP, Proteintech, Wuhan, Hubei, China), anti-Nectin-3 (11213-1-AP, Proteintech), 5-aza-2'-deoxycytidine (DAC, 189825, Sigma), Pierce™ Magnetic ChIP Kit (26157, Thermo, Waltham, MA, USA), Nano-Glo^® Dual-Luciferase^® Assay System (N1610, Promega), and puromycin (A1113802, Thermo) were purchased from the indicated manufacturers. The 5-week-old female BALB/c nude mice were obtained from the Medical Experimental Animal Center of Guangdong Province (Guangzhou, Guangdong, China). The microarray data used in this study were downloaded from the GEO database using the GSE52068 and GSE62336 accession numbers.

2.3 DAC treatment

An amount of 1.5 × 10⁵ NPC or NPE cells were seeded on 60 mm culture dishes (NEST Biotechnology, Wuxi, Jiangsu, China). After 24 h, the cells were treated with or without the methyltransferase inhibitor DAC (10 μmol/L) by replacing the drug every 24 h for 72 h. The cells were then collected for DNA and RNA extraction.

2.4 DNA extraction and bisulfite pyrosequencing

The genomic DNA was extracted from the NPC or NPE cells using the AllPrep RNA/DNA Mini kit (Qiagen, Dusseldorf, German) according to the manufacturer's instructions. The bisulfite transformation and purification of DNA (1-2 μg) were performed using the EpiTect Bisulfite Kit (Qiagen), following the manufacturer's instructions. The primers for polymerase chain reaction (PCR) and bisulfite pyrosequencing were designed by the PyroMark Assay Design Software 2.0 (Qiagen) and are listed in Supplementary Table S1. The PCR reactions were incubated at 95°C for 3 min, followed by 50 cycles of 15 s at 95°C, 20 s at 54°C and 30 s at 72°C, and a final 5 min hold at 72°C. The pyrophosphate sequencing reactions were conducted using the PyroMark Q96 ID system (Qiagen).

2.5 Real-time quantitative RT-PCR

RNA was extracted from NPE cells, NPC cells, and tissues using the TRIzol reagent (Invitrogen), and reverse transcription of the first-strand cDNA was performed using a reverse-transcription kit (Promega). The qRT-PCR assay was conducted on the Bio-Rad SFX (96 or 384) system with 2× SYBR Green mix (Life, Carlsbad, CA, USA). The data were normalized to the expression of GAPDH. The sequences of the primers are listed in Supplementary Table S1.

2.6 Lentivirus-mediated gene transfer

HEK293 cells were co-transfected with the indicated empty vector pLKO.1-shZNF582 or phage-6tag-ZNF582 and pSPAX2 and pMD2G (Addgene, Watertown, MA, USA). After 8-hour transfection, the culture medium was changed with fresh full medium. After an additional 8-hour transfection, the cell virus supernatants were harvested and used to infect SUNE1 or HONE1 cell lines. Then, 1 μg/mL puromycin was added to the medium to select the positive cells for 1-2 weeks. qRT-PCR and Western blotting assays were conducted to check the transfection efficiency.

2.7 Wound healing assay

SUNE1 or HONE1 cells were seeded in 6-well plates (NEST) to near confluence and then, incubated with serum-free medium for 24 h. Linear wounds were created using 200 μL tips in the cell monolayers, and the cells were washed twice with phosphate-buffered saline (PBS). The images were captured at 0 h and 24 h using a microscope (Leica, Wetzlar, Germany).

2.8 Migration and invasion assays

SUNE1, HONE1, NP69, or N2-Tert cells (5 × 10⁴ or 1 × 10⁵) were suspended in 200 μL of serum-free medium and seeded into upper Transwell chambers (8-μm pores, Corning, NY, USA) which were covered with (for invasion assay) or without matrigel (for migration assay, BD Biosciences). Then, the lower chambers were placed into 24-well plates (NEST) with a medium containing 10% FBS and incubated at 37°C. After 12 h (migration) or 24 h (invasion), these cells were fixed with methyl alcohol for 30 min and stained with crystal violet for 2 h. Migrated or invaded cells in 5 random visual fields were counted using a microscope (Leica).

2.9 Colony formation assay

SUNE1 (1000) or HONE1 cells (400) were seeded into a 6-well plate with full medium supplemented with 10% FBS, and cultured at 37°C for 1-2 weeks. Then, these cell colonies were fixed and stained with crystal violet. The images were captured using a microscope (Leica).

2.10 Cell adhesion assay

An empty 24-well plate with 10 μg/mL of fibronectin (Sigma) was incubated at 4°C overnight and washed thrice with PBS. Then, the plate was incubated with PBS containing 1% bovine serum albumin (BSA, Sigma) at 37°C for 1 h and washed thrice with PBS. Afterwards, SUNE1 or HONE1 cells (1 × 10⁵) were seeded into the 24-well plate supplemented with serum-free medium and incubated at 37°C for 1 h. The non-adhesion cells that contained the medium were aspirated off. Then, these cells were gently washed with PBS, fixed with methyl alcohol, and stained with crystal violet. The images were captured using a microscope (Leica).

2.11 Chromatin immunoprecipitation assays (ChIP)

A Pierce™ Magnetic ChIP Kit (26157, Thermo) was used to perform the ChIP assay according to the manufacturer's protocol. Briefly, the cells were fixed with 1% formaldehyde and quenched with glycine. Then, these cells were washed twice with ice-cold PBS, detached by scraping in ice-cold PBS with 10 μL of Halt Cocktail, and harvested in ChIP lysis buffer for 10 min on ice. Afterwards, the lysate was centrifuged (9000 × g for 3 min) at 4°C, and the nuclei were resuspended for MNase Digestion in a 37°C water bath for 15 min. The reaction was stopped using the MNase Stop Solution and centrifuged (9000 × g for 5 min) at 4°C. Next, 1× IP dilution buffer (Thermo) containing protease/phosphatase inhibitors was added to resuspend the nuclei. Then, the suspension was sonicated on ice with several pulses and incubated for 20 s on ice between pulses. The suspensions were centrifuged (9000 × g for 5 min) at 4°C, and the supernatant containing the digested chromatin was harvested. Afterwards, the supernatant was incubated with normal rabbit IgG, or the anti-FLAG or anti-RNA polymerase II antibody at 4°C overnight. Then, ChIP-grade protein G magnetic beads (25 μL) were added into the lysate in the following morning, and these were incubated at 4°C for 2 h with mixing. The DNA was eluted using 1X IP elution buffer (150 μL) containing 5 mol/L NaCl (6 μL) and 20 mg/mL proteinase K (2 μL) through incubation at 65°C for 30 min with vigorous shaking. The eluted DNA was purified using a DNA Clean-UP column and DNA column wash buffer. The purified DNA was used to perform the qRT-PCR (ChIP-qRT-PCR) detection or sequencing (ChIP-seq). The sequences for the ChIP-qRT-PCR primers are listed in Supplementary Table S1.

2.12 Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis

The above purified DNA was sequenced using the NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Then, the enriched genes (P < 0.05) were subjected to KEGG enrichment analysis by importing the list of genes into the DAVID website (https://david.ncifcrf.gov).

2.13 Reporter gene assay

SUNE1, HONE1, NP69, or N2-Tert cells (4 × 10⁴) were seeded in 24-well plates for 12 h and co-transfected with plasmids encoding empty vector or FLAG-ZNF582 or shZNF582, along with the pGL3-Nectin-3 or NRXN3 reporter plasmid (100 ng) and a phRL-TK-Renella luciferase control vector (Addgene, 10 ng). After 24 h, the cells were lysed in 1 × passive lysis buffer (Promega), and the luciferase activities were determined using the Nano-Glo^® Dual-Luciferase^® Assay System (Promega). Afterwards, the firefly luciferase activity was normalized through the Renella luciferase activity, and the results were presented as the fold change relative to the activity in the empty vector-transfected cells.

2.14 Inguinal lymph node metastasis models

SUNE1 cells (3 × 10⁵) stably encoding control shRNA (shControl) or shZNF582 were injected into the footpads of mice (n = 8 per group). These mice were euthanized after 35 days feeding, and the primary footpad tumors and linked inguinal lymph nodes were collected, fixed, paraffin-embedded, and sectioned. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) staining (anti-pan-cytokeratin antibody, Sigma) were conducted. The stained sections were captured using the AxioVision Rel.4.6 computerized image analysis system (Carl Zeiss, Jena, Germany). The popliteal lymph nodes with positive keratin IHC staining were considered as metastatic lymph nodes, and the metastatic popliteal lymph node ratio was defined as the proportion of metastatic lymph nodes among all resected popliteal lymph nodes.

2.15 In vivo xenograft tumor models

SUNE1 cells (1 × 10⁶) stably encoding empty vector or ZNF582 were injected into the tail vein of mice. The survival of mice was recorded every day. After 8 weeks, all mice were euthanized, and the lung tissues were fixed, paraffin-embedded, and sectioned, then, H&E or IHC staining was conducted. The stained sections were analyzed using a Leica O8 microscope. The intensities of the staining were analyzed using the ViewPointBETA v1 software (Portland, OR, USA).

2.16 Immunohistochemistry assay

NPE and NPC paraffin tissue sections and xenograft mice tissue sections were used to perform the IHC assay. Briefly, these tissues were deparaffinized at 60°C for 30 min and rehydrated. Then, the endogenous peroxidase was blocked using 3% H₂O₂ for 30 min, and the tissues were maintained at high-temperature citrate for 2.5 min for antigen retrieval. Subsequently, the non-specific binding proteins were blocked with BSA, and the tissues were incubated with the indicated primary antibodies at 4°C overnight. The staining of the paraffin sections was performed as previously described [31]. The staining intensity score was defined as follows: 0, no staining; 1, weak, light yellow; 2, moderate, yellow-brown; and 3, strong, brown. The positive rate score was defined as follows: 1, < 10%; 2, 10%-35%; 3, 35%-70%; 4, > 70%. The total score of indicated proteins was calculated as staining intensity score × positive rate score.

2.17 Statistical analysis

Unpaired two-tailed Student's t-test and one-way or two-way ANOVA analysis with the SPSS 20 software were used to compare the statistical differences among groups. All data are presented as mean ± standard deviation (SD) and were extracted from no less than three independent experiments. A P value of less than 0.05 was considered significant. The animal survival curves were generated using the Kaplan-Meier method and analyzed by the log-rank analysis.

3.1 The methylation level of ZNF582 promoter in NPC

We analyzed the methylation status of ZNF582 in the genome-wide DNA methylation microarray dataset (GSE52068) [9], and found that ZNF582 promoter was frequently hypermethylated in NPC tissues (Figure 1A). Six significantly hypermethylated CpG sites were identified in the ZNF582 prompter region (Figure 1B). The results were confirmed by microarray analysis with the same six CpG sites conducted in the Hong Kong dataset (GSE62336, Figure 1C). To validate the dysregulated methylation status of ZNF582 in NPC, we performed the bisulfite pyrosequencing against cg22647407 (the most significantly hypermethylated CpG site of ZNF582 ranked by P value, Figure 1D). The methylation level of ZNF582 was significantly higher in NPC tissues than in normal tissues (Figure 1E-F). Consistently, the ZNF582 methylation was elevated in NPC cell lines (Figure 1G). Together, these data demonstrated that ZNF582 was hypermethylated in NPC.

3.2 Association between ZNF582 hypermethylation and its expression in NPC

We next determined whether the hypermethylation of ZNF582 would affect its expression in NPC. qRT-PCR and Western blotting assays were conducted to evaluate the expression of ZNF582 in cell lines and tissue samples. Both mRNA and protein levels of ZNF582 were significantly decreased in NPC cell lines and tissues (Figure 2A-D), indicating that ZNF582 was down-regulated in NPC.

Next, we addressed whether the dysregulation of ZNF582 was caused by its promoter hypermethylation. After treatment with DAC, the ZNF582 methylation levels were significantly decreased in NPC cell lines (Figure 2E-F), whereas the mRNA levels of ZNF582 were substantially increased (Figure 2G), suggesting that ZNF582 was frequently down-regulated in NPC due to its promoter hypermethylation.

3.3 Association of ZNF582 with NPC cell migration and invasion in vitro

To determine the role of ZNF582 in the tumorigenesis or metastasis of NPC, colony formation and Transwell assays were performed with the NPC cell lines. The overexpression and two lentiviral shRNA constructs of ZNF582 were first generated, and the NPC cell lines with stable overexpression or knockdown of ZNF582 were established (Figure 3A-B). We found that the overexpression of ZNF582 significantly suppressed NPC cell migration and invasion (Figure 3C-D), while the knockdown of ZNF582 markedly increased the NPC cell migration and invasion (Figure 3E-F). However, neither the overexpression nor knockdown of ZNF582 had effect on NPC cell growth (data not shown). These results indicated that ZNF582 could inhibit NPC cell migration and invasion in vitro. To confirm these observations, we performed Transwell assays using normal nasopharyngeal epithelial cell lines (NP69 and N2-Tert) by knocking down ZNF582. Similar to the results in NPC cells, the knockdown of ZNF582 significantly increased the migration and invasion of NP69 and N2-Tert cells (Supplementary Figure S1). These results confirmed that ZNF582 inhibited the migration and invasion of both NPC and NPE cells.

3.4 Molecular mechanism of ZNF582 affecting NPC metastasis

Next, the mechanisms underlying the ZNF582-mediated suppressive effects in NPC were addressed. ChIP-seq against ZNF582 was performed to explore its downstream target genes, and a significant number of cell adhesion molecules were found to be highly enriched (Figure 4A). qRT-PCR confirmed that the adhesion molecule Nectin-3 was down-regulated (Figure 4B) and NRXN3 was up-regulated (Figure 4C) after ZNF582 overexpression in SUNE1 and HONE1 cells. Furthermore, after DAC treatment, the expression of ZNF582 was greatly increased in SUNE1 and HONE1 cells. Meanwhile, the expression of Nectin-3 was significantly inhibited and the expression of NRXN3 was greatly increased in SUNE1 and HONE1 cells (Supplementary Figure S2A), suggesting that ZNF582 could regulate the gene expression of Nectin-3 and NRXN3 at the mRNA level. To further validate these findings, we investigated the expression levels of Nectin-3 and NRXN3 in NP69 and N2-Tert cells. Compared with those in NP69 and N2-Tert cells, both the mRNA and protein levels of Nectin-3 were increased and NRXN3 were decreased in SUNE1 and HOEN1 cells (Supplementary Figure S2B-D). In addition, the knockdown of ZNF582 in NP69 or N2-Tert cells promoted the expression of Nectin-3 and inhibited the expression of NRXN3 (Supplementary Figure S2E-F). These results confirmed that ZNF582 could regulate the expression of Nectin-3 and NRXN3 on both mRNA and protein levels.

To assess how ZNF582 regulates the expression of Nectin-3 and NRXN3, ChIP-qPCR was performed. The binding motif of ZNF582 was predicted by previous CHIP-seq analysis, and one ZNF582-binding site in the Nectin-3 promoter and two ZNF582-binding sites in the NRXN3 promoter were identified (Figure 4D). The results revealed that ZNF582 could directly bind to the promoter regions of Nectin-3 and NRXN3 in NPC (Figure 4E-F) and NPE cells (Supplementary Figure S2G-H). To further confirm that ZNF582 drives the transcription of Nectin-3 and NRXN3, the upstream 1,000 bp (starting from the transcription starting site of Nectin-3 or NRXN3) was cloned to the pGL3 basic luciferase vector. Meanwhile, the constructs of Nectin-3 or NRXN3 promoter with mutations on the ZNF582-binding sites were made, and luciferase reporter assays were performed (Figure 4G). Consistent with previous qRT-PCR data in SUNE1 and HONE1 cells, ZNF582 was found to potently inhibit the luciferase activity of the wild-type Nectin-3 promoter, but not the mutant Nectin-3 promoter (Figure 4H). Meanwhile, ZNF582 significantly increased the luciferase activity of the wild-type NRXN3 promoter, but not the mutant NRXN3 promoter (Figure 4I). Furthermore, the knockdown of ZNF582 in NP69 and N2-Tert cells demonstrated opposite effects (Supplementary Figure S2I-J). Together, these data suggested that ZNF582 could directly bind to the promoter of Nectin-3 or/and NRXN3 and drove the transcriptional inhibition or activation of these genes.

3.5 Association of Nectin-3 with NPC cell adhesion, migration, and invasion in vitro

We analyzed the expression of Nectin-3 in NPE and NPC tissues archived in the GSE12452 dataset and found that Nectin-3 was significantly overexpressed in NPC tissue samples (Figure 5A) and was inversely correlated with ZNF582 expression (R = -0.310, P = 0.049) (Figure 5B), suggesting that Nectin-3 could be an oncogene in NPC. To identify the exact role of Nectin-3 in NPC, the adhesion, migration, and invasion abilities of NPC cells with the overexpression or knockdown of Nectin-3 were detected. The overexpression of Nectin-3 promoted the cell adhesion, migration, and invasion of NPC cells (Figure 5C-D), while the knockdown of Nectin-3 inhibited these abilities (Figure 5E). These results indicated that Nectin-3 acts as an oncogene in NPC.

3.6 Association of NRXN3 with NPC cell adhesion, migration, and invasion in vitro

NRXN3 expression was lower in NPC tissues than in normal tissues (Figure 5F) and was positively correlated with ZNF582 expression in NPC tissues (GSE12452) (Figure 5G; R = 0.734, P < 0.001). We hypothesized that NRXN3 could function as a tumor suppressor gene in NPC. Cell adhesion, migration, and invasion assays were performed to identify the role of NRXN3 in NPC. As expected, the knockdown of NRXN3 increased the adhesion, migration, and invasion abilities of NPC cells (Figure 5H-I), indicating that NRXN3 acted as a tumor suppressor in NPC.

3.7 Both Nectin-3 and NRXN3 are functional targets of ZNF582 in NPC

We confirmed that ZNF582 induced the down-regulation of Nectin-3 by inhibiting its transcription. Hence, we determined the effects of Nectin-3 and NRXN3 on the tumor repressor function of ZNF582 in vitro. The overexpression of Nectin-3 (Figure 6A-B) and knockdown of NRXN3 (Figure 6C-D) reversed the suppressive effect of ZNF582 on the adhesion, migration, and invasion of NPC cells that stably overexpressed ZNF582, suggesting that both Nectin-3 and NRXN3 were functional targets of ZNF582 in NPC.

3.8 ZNF582 inhibits NPC cell invasion and metastasis in vivo

To validate the functions of ZNF582 in NPC in vivo, a popliteal lymph node metastasis model was constructed by transplanting SUNE1 cells stably expressing shZNF582 or shControl into the footpads of nude mice. After 35 days, the primary tumors and popliteal lymph nodes were obtained for analysis (Figure 7A). H&E staining revealed that the primary tumors in the ZNF582 knockdown group had a more aggressive phenotype with the invasion of tumor cells towards the skin and muscle, when compared to tumors in the shControl group (Figure 7B). Furthermore, the volume of the popliteal lymph nodes and the number of pan-cytokeratin-positive tumor cells were greater in the ZNF582 knockdown group than in the shControl group (Figure 7C-D). Strikingly, the metastatic popliteal lymph node ratio was remarkably higher in the ZNF582 knockdown group than in the shControl group (Figure 7E; P < 0.05).

To further investigate the effect of ZNF582 on NPC metastasis in vivo, lung metastasis models were employed. Mice injected with SUNE1 that stably restored ZNF582 exhibited significantly reduced metastatic tumor colonies and tumor density in the lungs (Figure 7F-I) and longer survival (Figure 7J) when compared to mice injected with control cells, suggesting that ZNF582 could suppress NPC cell metastasis in vivo.

To further determine whether ZNF582 regulates the expression of Nectin-3 and NRXN3 in vivo, IHC staining was performed to detect the expression of these proteins in lung metastasis. Consistent with the present in vitro findings, the expression of Nectin-3 was significantly decreased and the expression of NRXN3 was significantly increased in the ZNF582 overexpression group, when compared with those in the vector group (Figure 7K). Moreover, the same results were obtained for the expression levels of Nectin-3 and NRXN3 in NPE and NPC tissue samples (Figure 7L-M). These results indicated that the up-regulation of ZNF582 reduced the expression of Nectin-3 and increased the expression of NRXN3 in vivo.