ZNF844 Suppresses Invasion and Metastasis in Nasopharyngeal Carcinoma by Inhibiting the PI3K‐AKT Signaling Pathway

ABSTRACT

Epigenetic regulation plays a crucial role in the development of nasopharyngeal carcinoma (NPC). However, the epigenetic mechanisms underlying NPC recurrence and metastasis remain poorly understood. This study aimed to investigate and identify a novel molecular target with prognostic relevance in NPC. The methylation status of zinc finger protein 844 (ZNF844) was assessed in NPC tissues and cell lines using bisulfite pyrosequencing. ZNF844 expression levels in NPC cell lines and clinical specimens were analyzed by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) and Western blotting. Stable NPC cell lines with ZNF844 overexpression or knockdown were established to evaluate its biological functions both in vitro and in vivo. RNA sequencing was performed to identify downstream targets of ZNF844, followed by validation using qRT‐PCR and Western blotting. We found that ZNF844 expression was significantly downregulated in NPC tissues and cell lines, correlating with hypermethylation of its promoter region. Treatment with the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine (DAC) restored ZNF844 mRNA expression. ZNF844 overexpression suppressed NPC cell invasion and metastasis, whereas its silencing had the opposite effect. In xenograft models, ZNF844 overexpression reduced metastatic burden. ZNF844 inhibited the PI3K‐AKT signaling pathway, thereby suppressing NPC cell invasiveness. Clinically, high ZNF844 expression was associated with improved overall survival and distant metastasis‐free survival. ZNF844 inhibits the invasive and metastatic capabilities of nasopharyngeal carcinoma by modulating the PI3K–AKT signaling pathway. These inhibitory effects can be partially or completely reversed by treatment with SC79. It may serve as a promising therapeutic target and prognostic biomarker for NPC.

ZNF844 functions as a tumor suppressor in NPC by inhibiting invasion and metastasis through regulation of the PI3K‐AKT signaling pathway. It may serve as a promising therapeutic target and prognostic biomarker for NPC.

Abbreviations

ccRCC

clear‐cell renal cell carcinoma

DAC

5‐aza‐2'‐deoxycytidine

DMFS

distant metastasis‐free survival

H&E

hematoxylin and eosin

HNSCC

head and neck squamous cell carcinoma

IHC

immunohistochemistry

NPC

nasopharyngeal carcinoma

NPE

nasopharyngeal epithelial

OS

overall survival

qRT‐PCR

quantitative reverse transcription‐polymerase chain reaction

ZNF844

zinc finger protein 844

ZFPs

zinc finger proteins

1. Introduction

Nasopharyngeal carcinoma (NPC) is a rare malignant tumor with a distinct geographic distribution, showing particularly high prevalence in southern China and Southeast Asia [1, 2]. Because the nasopharynx is anatomically concealed and the tumor is highly sensitive to radiotherapy, the standard treatment for NPC is radiotherapy, either alone or combined with chemotherapy [3]. Over the past few decades, advances in molecular biology, imaging, and therapeutic strategies have significantly reduced morbidity and mortality. Nevertheless, recurrence and distant metastasis remain the principal causes of treatment failure in patients with locally advanced NPC [4, 5]. This underscores the urgent need to elucidate the molecular mechanisms driving NPC progression and to identify reliable biomarkers for early diagnosis, prognostic assessment, and the development of novel targeted therapies.

DNA methylation, the most common and earliest recognized epigenetic modification, plays a pivotal role in maintaining genomic stability and regulating essential cellular processes such as cell cycle progression, apoptosis, and embryonic development [6, 7, 8]. Aberrant methylation of specific genes has been implicated in promoting tumor cell proliferation, invasion, and migration, thereby contributing to tumor progression and recurrence [9].

Transcription factors are central regulators of gene expression and participate in a wide range of physiological functions, including differentiation, development, metabolism, apoptosis, and autophagy [10]. Dysregulation of transcription factor expression is a recognized driver of tumorigenesis. In eukaryotes, zinc finger proteins (ZFPs) represent the largest family of transcription factors, and accumulating evidence has demonstrated their critical involvement in cancer pathogenesis. For example, Zhao et al. reported that hypermethylation of ZNF582 suppresses its expression in NPC and that ZNF582 overexpression inhibits NPC cell proliferation, migration, invasion, and metastasis both in vitro and in vivo [11]. Similarly, ZNF671 is frequently hypermethylated, leading to reduced expression, S‐phase arrest inhibition, and enhanced NPC cell proliferation through downregulation of p21 and upregulation of cyclin D1 and c‐myc [12]. ZNF382 has also been shown to inhibit NPC cell proliferation and oncogene expression by suppressing NF‐κB and AP‐1 signaling pathways, whereas promoter methylation silences ZNF382 and activates these oncogenic cascades [13].

In a previous genome‐wide methylation microarray analysis (GSE52068) of 24 normal and 24 NPC tissues, we were the first to identify hypermethylation of the transcription factor zinc finger protein 844 (ZNF844) in nasopharyngeal carcinoma [14]. ZNF844 expression is downregulated across multiple tumor types, including head and neck cancer, renal carcinoma, breast cancer, and lung cancer, and its aberrant expression correlates with patient survival in clear‐cell renal cell carcinoma (ccRCC) and head and neck squamous cell carcinoma (HNSCC) [15]. Despite these associations, the biological role and mechanistic function of ZNF844 in NPC remain largely unknown.

Based on these findings, the present study aimed to elucidate the role of ZNF844 in NPC invasion and metastasis and to explore the underlying molecular mechanisms, with the goal of assessing its potential as a prognostic biomarker and therapeutic target.

2. Materials and Methods

2.1. Clinical Specimens and Cell Culture

A total of 126 NPC tissues were obtained from Wuzhou Red Cross Hospital, and 11 normal nasopharyngeal epithelial (NPE) tissues were collected from the Affiliated Hospital of Guilin Medical University. Patients with NPC received radiotherapy combined with cisplatin‐based chemotherapy. Induction chemotherapy regimens included the TPF regimen (docetaxel, cisplatin, and 5‐fluorouracil) and the PF regimen (cisplatin and 5‐fluorouracil). Cisplatin was employed in concurrent chemotherapy. The PF regimen (cisplatin and 5‐fluorouracil) was used in adjuvant chemotherapy. All patients received intensity‐modulated radiation therapy (IMRT). The prescribed dose to the gross tumor volume (GTV) exceeded 66 Gy, and > 50 Gy was delivered to bilateral cervical lymph nodes and potential sites of microscopic infiltration. These samples were used for bisulfite pyrosequencing of the ZNF844 promoter region and for immunohistochemical (IHC) analysis of ZNF844 protein expression to assess patient prognosis. Besides, the six normal nasopharyngeal epithelial tissues and NPC samples used for promoter methylation analysis and mRNA expression analysis were obtained from the same sample cohort.

NPC cell lines (5‐8F, CNE2, HNE1, HONE1, CNE1, S18, 6‐10B, and SUNE1), the immortalized N2‐Tert cell line, and the 293 T cell line were obtained from the Sun Yat‐sen University Cancer Center (Guangzhou, China). NPC cell lines were cultured in RPMI‐1640 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; ThermoFisher Scientific). N2‐Tert cells were maintained in keratinocyte serum‐free medium (KSFM; Invitrogen, Carlsbad, CA, USA) containing bovine pituitary extract (BD Biosciences, Franklin Lakes, NJ, USA). 293 T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sangon Biotech, Shanghai, China) supplemented with 10% FBS. All cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

2.2. 5‐Aza‐2′‐Deoxycytidine Treatment

Log‐phase NPC cells were seeded in 6‐well plates (1 × 10⁵ cells/well) and treated with 10 μM 5‐aza‐2′‐deoxycytidine (DAC; Sigma‐Aldrich, Steinheim am Albuch, Germany) for 72 h, with fresh medium containing DAC replaced every 24 h. Cells were then harvested for DNA and RNA extraction.

2.3. DNA Extraction and Bisulfite Pyrosequencing

Genomic DNA was extracted from tissues and cell lines (with or without DAC treatment) using the AllPrep RNA/DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Bisulfite conversion was performed using the EpiTect Bisulfite Kit (Qiagen). Primers for ZNF844 were designed using PyroMark Assay Design 2.0 (Qiagen). Primer sequences are provided in Supplementary Table S1. Methylation levels at each CpG site were quantified using the Pyro Q‐CpG software (Qiagen).

2.4. Real‐Time Quantitative RT‐PCR

Total RNA was extracted using TRIzol reagent (Tiangen, Beijing, China), and 2 μg of total RNA was reverse‐transcribed according to the manufacturer's instructions. The reverse transcription system and procedure are detailed in Supplementary Table S1. qRT‐PCR was performed using gene‐specific primers (Supplementary Table S1, Supplementary Table S1) and SYBR Green Master Mix (Thermo Fisher Scientific) on an ABI 7500 Real‐Time PCR System (Applied Biosystems, Carlsbad, CA, USA). GAPDH was used as the internal control, and relative expression levels were calculated using the 2^⁻ΔΔCt method.

2.5. Western Blotting Assays

Cells and tissues were lysed in RIPA buffer (Solarbio, Beijing China) containing protease and phosphatase inhibitors. Lysates were sonicated at 25 Hz, incubated on ice for 30 min, and centrifuged at 12,000 rpm for 30 min at 4°C. Protein concentrations were determined using the BCA Protein Assay Kit (Invitrogen). Equal amounts of protein (30 μg) were separated by SDS‐PAGE (Beyotime, Shanghai, China) and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% skim milk or 5% bovine serum albumin (BSA) for 2 h at room temperature, incubated overnight at 4°C with primary antibodies, and then with fluorescent secondary antibodies for 1 h at room temperature. Signals were visualized using the ODYSSEY dual‐color infrared fluorescence imaging system (LI‐COR Biosciences, Lincoln, NE, USA).

2.6. Vector Constructure, Cell Transfection, and Lentiviral Infection

For ZNF844 overexpression, the Psin‐EF2‐HA‐Puro‐ZNF844 plasmid was purchased from LongBio (Guangzhou, China). For gene silencing, siRNA targeting ZNF844 (siZNF844) was obtained from Hanheng Biotechnology (Shanghai, China) (Supplementary Table S1). Transient transfections were performed using Lipofectamine 3000 (Invitrogen). For stable cell line generation, Psin‐EF2‐HA‐Puro or Psin‐EF2‐HA‐Puro‐ZNF844 plasmids were co‐transfected with packaging plasmids pSPAX2 and pMD2.G into 293 T cells. Viral supernatants were collected 48 h post‐transfection, filtered, and used to infect 5‐8F and CNE2 cells. Stable clones were selected using puromycin (2 μg/mL) and verified by qRT‐PCR and Western blotting.

2.7. Wound Healing Assay

5‐8F and CNE 2 cells transfected with empty vector or overexpressing ZNF844 were inoculated into 6‐well plates. When the cell confluence reached 90%, the monolayer of cells was mechanically scratched from top to bottom using a 10‐μL pipette tip with the same strength. Cells were washed with PBS and cultured in serum‐free medium. Images were taken at 0 h and 24 h under an inverted microscope (×4).

2.8. Transwell Migration and Invasion Assays

Cell migration and invasion were assessed using Transwell chambers (8 μm pore size; Corning, Corning, NY, USA) with or without Matrigel coating (BD Biosciences). A total of 5 × 10⁴ cells in 200 μL serum‐free medium were seeded into the upper chamber, and 600 μL medium containing 20% FBS was added to the lower chamber. After incubation (12 h for migration, 24 h for invasion) at 37°C, cells were fixed with 4% paraformaldehyde for 1 h and stained with 0.1% crystal violet for 15 min. The number of migrated or invaded cells was then observed and photographed under an inverted microscope at 10× magnification.

2.9. RNA Sequencing and Analysis

Cells transfected with ZNF844 or an empty vector were harvested, and total cellular RNA was extracted using TRIzolL reagent. RNA integrity and DNA contamination were analyzed by agarose gel electrophoresis. RNA purity (OD260/230 ratio) was detected by a NanoPhotometer spectrophotometer (GmbH Munich, Germany), while RNA Integrity Number (RIN) was accurately measured by an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, US).

Eukaryotic mRNA was enriched using magnetic beads with Oligo(dT) (Thermo Fisher Scientific) and then fragmented. A first‐strand cDNA was synthesized from the mRNA template using random hexamers, followed by second‐strand cDNA synthesis. The resulting double‐stranded cDNA was purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA) before undergoing end repair, A‐tailing, and adapter ligation. After fragment size selection with AMPure XP beads, the final library was obtained through PCR amplification and subsequent purification with AMPure XP beads. Following quality control, the libraries were pooled according to their effective concentration and desired data volume. The pooled libraries were loaded onto an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) for high‐throughput sequencing.

2.10. In Vivo Tumorigenicity and Lung Metastasis

Male BALB/c‐nude mice (4–6 weeks old) were purchased from Jiangsu Huasino Pharmaceutical Technology Co Ltd. (Lianyungang, Jiangsu, China) and maintained under pathogen‐free conditions. The effect of ZNF844 on the tumorigenic and metastatic potential of NPC cells was analyzed using subcutaneous and systemic metastasis in vivo models. For the subcutaneous model, 1 × 10⁶ empty vector‐ or ZNF844‐overexpressing 5‐8F cells, mixed with Matrigel (BD Biosciences), were subcutaneously injected into the right flank of each mouse. After 30 days, the mice were euthanized, and the subcutaneous tumors were harvested for hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining. For the lung metastasis model, 1 × 10⁶ empty vector‐ or ZNF844‐overexpressing 5‐8F cells were injected directly into the tail vein. The mice were euthanized 60 days after inoculation, and sections of the lung were harvested and analyzed using H&E and IHC staining.

2.11. Hematoxylin–Eosin Staining Assays and Immunohistochemistry

The subcutaneous tumor tissues or lung tissues were fixed in 4% paraformaldehyde, paraffin‐embedded, and sectioned at 4 μm. All NPC tissue specimens included in this study were fixed in a sufficient volume of 10% neutral‐buffered formalin within 30 min after surgical resection. Sections were stained with H&E or subjected to IHC for ZNF844 expression. Antigen retrieval was performed in EDTA buffer, and endogenous peroxidase was quenched with 3% hydrogen peroxide. Sections were blocked with goat serum and incubated overnight at 4°C with anti‐ZNF844 antibody (CUSABIO, China), followed by biotin‐labeled secondary antibody and DAB (Cell Signaling Technology, Danvers, MA, USA) visualization. The staining results were assessed using a semi‐quantitative scoring system. Staining intensity was graded as follows: 0 for no detectable positive signal, 1 for pale yellow (weak positivity), 2 for brown–yellow (moderate positivity), and 3 for dark brown (strong positivity). Concurrently, the proportion of positive cells was categorized into four levels: 1 (less than 25% positive cells), 2 (26%–50%), 3 (51%–75%), and 4 (greater than 75%). The final immunohistochemical score was determined by multiplying the intensity score by the extent score. All tissue sections were independently evaluated by two experienced pathologists under double‐blind conditions. Immunohistochemical scoring data were analyzed using X‐tile software (Rimm Lab at Yale University, New Haven, CT, USA), and the optimal cut‐off value for prognostic stratification was identified as 4 based on the minimum p‐value approach. Based on this threshold, patients were classified into two groups: the high ZNF844 expression group and the low ZNF844 expression group.

2.12. Statistical Analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using SPSS 28.0 (IBM, Armonk, NY, USA) and GraphPad Prism 9.5 (GraphPad Software, Boston, MA, USA). Differences between groups were assessed using unpaired Student's t‐test or χ² test, as appropriate. Survival curves were generated by the Kaplan–Meier method and compared using the log‐rank test. p < 0.05 was considered statistically significant.

3. Results

3.1. The ZNF844 Promoter Is Hypermethylated in NPC

Our previous genome‐wide DNA methylation microarray data (GSE52068) on 24 pairs of NPC and normal nasopharyngeal tissues revealed that CpG sites in the ZNF844 gene were hypermethylated in NPC tissues but not in normal nasopharyngeal tissues (Figure 1A,B). To validate this finding, we performed bisulfite pyrosequencing on a specific promoter region, from –228 to −147 bp upstream of the transcription start site. This analysis confirmed that the methylation level of the ZNF844 promoter region was consistently higher in NPC cells and tissues than in normal cells and tissues (Figure 1C–F).

FIGURE 1.

ZNF844 promoter region is hypermethylated in NPC. (A) Heatmap clustering analysis of CpG (cytosine–guanine) sites in the ZNF844 gene comparing NPC tissues (n = 24) with normal nasopharyngeal tissues (n = 24) from dataset GSE52068. (B) Methylation levels of 10 CpG sites of ZNF844 in normal and NPC tissues in dataset GSE52068. (C) Schematic representation of CpG islands and the bisulfite pyrosequencing region within the ZNF844 promoter. Red region: Input sequence; blue region: CpG island; TSS: Transcription start site; red text: CpG site analyzed by bisulfite sequencing. (D–E) Methylation levels of the ZNF844 promoter in normal nasopharyngeal tissues (n = 6) and NPC tissues (n = 6). (F) Methylation levels in N2‐Tert cells and NPC cell lines (HONE1, SUNE1, HNE1, CNE1, CNE2, 5‐8F, 6‐10B, and S18). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

3.2. Hypermethylation‐Mediated Downregulation of ZNF844 in NPC

We next determined whether the hypermethylation of ZNF844 affects its expression in NPC. RT‐qPCR analysis confirmed that the expression of ZNF844 mRNA was significantly lower in NPC cell lines and tissues compared to N2‐Tert cells and normal nasopharyngeal tissues (Figure 2A,B). Consistent with these findings, the protein levels of ZNF844 were also lower in NPC cell lines and tissues (Figure 2C–F). To investigate if ZNF844 expression is regulated by its methylation status, we treated N2‐Tert and NPC cells with the DNA methyltransferase inhibitor DAC. RT‐qPCR assays showed that DAC was able to downregulate the methylation of the ZNF844 promoter and upregulate its expression in NPC cells (Figure 2G,H). Therefore, these data demonstrate that promoter DNA hypermethylation of ZNF844 is likely responsible for the downregulation of its expression.

FIGURE 2.

ZNF844 promoter hypermethylation downregulates its expression in NPC. (A) Quantitative RT–PCR analysis of ZNF844 mRNA expression in N2‐Tert and NPC cell lines. (B) ZNF844 mRNA expression in six normal nasopharyngeal epithelial tissues and 16 NPC tissues. (C–F) Western blot analysis of ZNF844 expression in N2‐Tert cells and NPC cell lines (HONE1, SUNE1, HNE1, CNE1, CNE2, 5‐8F, S18, and 6‐10B) and in normal nasopharyngeal epithelial tissues (N, n = 5) and NPC tissues (T, n = 5). (G) Comparison of ZNF844 promoter methylation levels in N2‐Tert and NPC cell lines with or without 5‐aza‐2′‐deoxycytidine (DAC) treatment by bisulfite sequencing. (H) ZNF844 mRNA expression in N2‐Tert and NPC cell lines with or without DAC treatment, analyzed by RT–qPCR. *p < 0.05; ****p < 0.0001.

3.3. ZNF844 Inhibits NPC Cell Migration and Invasion In Vitro

To explore the role of ZNF844 in NPC tumorigenesis, NPC cell lines stably overexpressing ZNF844 or transiently transfected with siRNA targeting ZNF844 (siZNF844) were established (Figure 3A–C, Figure 4A–C). Transwell assays revealed that ZNF844 overexpression significantly suppressed cell migration and invasion in 5‐8F and CNE2 cells (Figure 3D,E), whereas ZNF844 knockdown enhanced these abilities (Figure 4D–G). Wound healing assays further confirmed that ZNF844 overexpression impaired NPC cell migratory capacity (Figure 3F). These findings suggest that ZNF844 functions as a negative regulator of NPC cell motility.

FIGURE 3.

Overexpression of ZNF844 inhibits NPC cell migration and invasion in vitro. (A) RT–qPCR analysis of ZNF844 mRNA expression in 5‐8F and CNE2 cells transfected with the empty vector or ZNF844 overexpression plasmid. (B,C) Western blot analysis of ZNF844 level in 5‐8F and CNE2 cells stably overexpressing ZNF844. (D) Transwell migration assay (without Matrigel) showing reduced migration of 5‐8F and CNE2 cells overexpressing ZNF844. (E) Transwell invasion assay (with Matrigel) showing reduced invasion of 5‐8F and CNE2 cells overexpressing ZNF844. (F) Wound healing assay demonstrating reduced migration of 5‐8F and CNE2 cells overexpressing ZNF844. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4.

Knockdown of ZNF844 promotes invasion and metastasis in vitro. (A) RT–qPCR analysis of ZNF844 mRNA expression in 5‐8F and CNE2 cells transfected with siNC or siZNF844. (B,C) Western blot analysis confirming ZNF844 knockdown in 5‐8F and CNE2 cells. (D,E) Transwell migration assay (without Matrigel) showing increased migration in 5‐8F and CNE2 cells after ZNF844 knockdown. (F,G) Transwell invasion assay (with Matrigel) showing increased invasion in 5‐8F and CNE2 cells after ZNF844 knockdown. *p < 0.05; **p < 0.01; ***p < 0.001.

3.4. ZNF844 Suppresses the PI3K‐AKT Signaling Pathway

RNA sequencing followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses identified the PI3K–AKT pathway as one of the most significantly altered signaling cascades upon ZNF844 overexpression (Figure 5A,B). Western blotting demonstrated that ZNF844 overexpression reduced phosphorylation levels of PI3K and AKT in both 5‐8F and CNE2 cells. Following treatment with SC79, an activator of AKT, the inhibitory effect of ZNF844 on p‐PI3K and p‐AKT expression was markedly reversed (Figure 5D,F). Conversely, ZNF844 knockdown increased phosphorylation of PI3K and AKT (Figure 5E,G), indicating that ZNF844 inhibits NPC invasion and metastasis, at least in part, by suppressing the PI3K–AKT signaling pathway (Figure 5C).

FIGURE 5.

Differential gene enrichment in the PI3K‐AKT pathway following ZNF844 overexpression. (A) Union heatmap of gene expression clustering. Each row represents a gene, and each column represents a sample; genes with similar expression values are clustered at both the row and column levels. (B) Top 20 KEGG‐enriched signaling pathways identified after stable ZNF844 overexpression in 5‐8F cells. (D and F) Western blot analysis revealed the levels of phosphorylated PI3K and phosphorylated AKT proteins in 5‐8F and CNE2 cells across different treatment groups. (E and G) Increased phosphorylated PI3K and phosphorylated AKT protein levels in 5‐8F and CNE2 cells after ZNF844 knockdown. (C) Schematic illustration of the proposed mechanism by which ZNF844 suppresses NPC invasion and metastasis via the PI3K–AKT pathway.

In the rescue experiments, wound healing and Transwell assays demonstrated that the invasive and migratory capacities of nasopharyngeal carcinoma cells in the ZNF844‐overexpressing group were significantly reduced compared with those in the vector group. Treatment with SC79 effectively reversed the inhibitory effect of ZNF844 overexpression on cell migration and invasion (Figure 6A–F). Collectively, these findings indicate that activation of the PI3K–AKT signaling pathway by SC79 can counteract the suppressive effects of ZNF844 overexpression on the in vitro progression of NPC cells.

FIGURE 6.

Overexpression of ZNF844 inhibits cell migration and invasion ability by blocking the PI3K‐AKT signaling pathway. (A‐D) The migratory and invasive capabilities of 5‐8F and CNE2 cells in the vector group, ZNF844‐OE group, and SC79 treatment group were assessed using Transwell assays. (E,F) The migratory capabilities of 5‐8F and CNE2 cells in different treatment groups were assessed by means of wound healing assays. *p < 0.05; **p < 0.01; ***p < 0.001.

3.5. ZNF844 Attenuates Tumor Growth and Metastasis In Vivo

In a subcutaneous xenograft model, tumors derived from ZNF844‐overexpressing 5‐8F cells were significantly smaller and lighter than those from control cells (Figure 7A–C). IHC confirmed higher ZNF844 protein expression in the overexpression group (Figure 7B). In a lung metastasis model, mice injected with ZNF844‐overexpressing cells exhibited markedly fewer metastatic nodules compared with controls (Figure 7D,E–G). IHC and H&E staining confirmed reduced metastatic burden in the lungs (Figure 7F,G).

FIGURE 7.

ZNF844 inhibits tumor growth and metastasis in vivo. (A) Images of excised subcutaneous tumors. (B) Immunohistochemical staining showing ZNF844 protein expression in subcutaneous tumor tissues. (C) Tumor weights in mice injected with ZNF844‐overexpressing or control 5‐8F cells. (D,E) Representative images of lung metastases in the nude mouse model; red arrows indicate metastatic lesions. Comparison of lung metastasis counts between ZNF844‐overexpressing and control groups. (F) Immunohistochemical staining of ZNF844 expression in lung metastases. (G) Hematoxylin–eosin staining showing fewer lung metastases in the ZNF844‐overexpressing group compared with controls. **p < 0.01; ***p < 0.001.

3.6. Low ZNF844 Expression Correlates With Poor Clinical Outcome in NPC

IHC analysis of NPC specimens from 105 patients revealed that low ZNF844 expression was significantly associated with inferior overall survival (OS) and distant metastasis‐free survival (DMFS) (Figure 8A–D). Kaplan–Meier analysis demonstrated that patients with high ZNF844 expression had significantly better OS and DMFS than those with low expression (log‐rank test, p < 0.05). Clinicopathological correlations are summarized in Table 1.

FIGURE 8.

Prognostic analysis of ZNF844 expression in NPC by immunohistochemistry. (A,B) Representative immunohistochemical staining of NPC patient samples from the ZNF844 low‐expression group (A) and high‐expression group (B). (C) Kaplan–Meier curves comparing overall survival (OS) between ZNF844 high‐ and low‐expression groups. (D) Kaplan–Meier curves comparing distant metastasis‐free survival (DMFS) between ZNF844 high‐ and low‐expression groups.

TABLE 1.

Relationship between ZNF844 expression and clinical characteristics of NPC (n = 105).

Clinical characteristics	ZNF844‐high group (n, %)	ZNF844‐low group (n, %)	P value
Age (years)
≤ 47 years	22 (52.4)	31 (49.2)	0.750
> 47 years	20 (47.6)	32 (50.8)
Sex
Male	28 (66.7)	49 (77.8)	0.207
Female	14 (33.3)	14 (22.2)
T stage
T1 T2 T3 T4	5 (11.9) 10 (23.8) 11 (26.2) 16 (38.1)	8 (12.7) 12 (19.0) 17 (27.0) 26 (41.3)	0.949
N stage
N0 N1 N2 N3	1 (2.4) 5 (11.9) 28 (66.7) 8 (19.0)	3 (4.8) 8 (12.7) 49 (77.8) 3 (4.8)	0.126
TNM stage
II III IVa IV b	3 (7.1) 21(50.0) 10(23.8) 8(19.0)	6(9.5) 30(47.6) 24(38.1) 3(4.8)	0.082
Inducing chemotherapy
Yes No	22(52.4) 20(47.6)	21(33.3) 42(66.7)	0.052
Concurrent chemotherapy
Yes No	37(88.1) 5(11.9)	55(87.3) 8(12.7)	0.904
Adjuvant chemotherapy
Yes No	8(19.0) 34(81.0)	7(11.1) 56(88.9)	0.255

Note: The p‐value was determined using χ2 tests.

Abbreviations: NPC, nasopharyngeal carcinoma; TNM, tumor‐node‐metastasis.

4. Discussions

This study demonstrates that ZNF844 expression is markedly reduced in NPC, largely due to promoter hypermethylation. Functional analyses revealed that ZNF844 acts as a tumor suppressor, inhibiting migration and invasion in vitro and reducing tumor growth and metastasis in vivo. ZNF844 appears to exert its anti‐metastatic effects by suppressing the PI3K–AKT signaling pathway. These findings identify ZNF844 as a potential prognostic biomarker and therapeutic target in NPC.

KRAB‐zinc finger proteins (KRAB‐ZFPs) are the largest family of mammalian transcription regulators. Members of this family, such as ZNF844, are increasingly recognized for their roles in tumor initiation, progression, and immune modulation. Evidence from multiple cancer types highlights ZNF844's significance as a potential prognostic biomarker and tumor suppressor. In ccRCC, reduced ZNF844 expression has been correlated with advanced stage, high grade, presence of metastasis, and poor prognosis and has been suggested as an independent prognostic factor [15]. Similar trends have been reported in HNSCC, where low ZNF844 expression predicts unfavorable survival outcomes [15]. Whole‐exome sequencing studies in breast cancer have identified ZNF844 mutations among key cancer driver genes, supporting its relevance in tumorigenesis [16]. According to the IntOGen and CGC databases, alterations in ZNF844 are implicated in multiple malignancies, including cervical cancer, colorectal adenocarcinoma, and ovarian cancer [16]. Beyond tumor growth regulation, ZNF844 may also influence the tumor immune microenvironment. Previous studies have linked its expression to infiltration of Th1 cells, suggesting a role in promoting antitumor immunity [15, 17]. Our in vitro and in vivo results align with this notion, showing that ZNF844 overexpression suppresses NPC aggressiveness, consistent with its tumor‐suppressive role in ccRCC.

Transcriptome profiling in this study revealed that the PI3K–AKT pathway was among the most significantly downregulated pathways upon ZNF844 overexpression. Furthermore, this study represents the first report demonstrating that ZNF844 functions as a regulator of the PI3K/AKT pathway and plays a critical role in modulating the invasion and metastasis of nasopharyngeal carcinoma cells. This pathway is central to regulating cell proliferation, survival, metabolism, and migration [18, 19, 20]. Dysregulation of PI3K–AKT signaling is a common event in tumor progression and metastasis [21, 22, 23]. Numerous zinc finger protein‐coding genes have been shown to exert tumor suppressor effects through modulation of the PI3K/AKT signaling pathway [24, 25], thereby downregulating the expression levels of p‐PI3K and p‐AKT. In the present study, overexpression of ZNF844 significantly reduced the expressions of p‐PI3K and p‐AKT and suppressed the migration and invasion of NPC cells. These findings were consistent with those of previous studies, which further suggested that the PI3K/AKT signaling pathway may be blocked by ZNF844 in NPC. Rescue experiments reversed the inhibitory effects of ZNF844 overexpression on the migration and invasion of NPC cells. Future studies could include animal experiments to verify whether ZNF844 inhibits tumor growth in vivo upon suppression of the PI3K/AKT signaling pathway, further supporting its functional role through this pathway. Overall, additional research is required to establish a more comprehensive theoretical foundation and provide guidance for the clinical translation of therapeutic targets in nasopharyngeal carcinoma.

Taken together, our findings add to the growing evidence that epigenetic silencing of tumor suppressor genes contributes to NPC progression. By demonstrating that ZNF844 downregulation via promoter hypermethylation promotes invasion and metastasis through PI3K–AKT activation, we provide a mechanistic basis for its prognostic value. Future research is needed to determine if the therapeutic restoration of ZNF844 expression, potentially using demethylating agents, could improve clinical outcomes in NPC.

ZNF844 functions as a tumor suppressor in NPC, with its expression frequently silenced by promoter hypermethylation. Restoration of ZNF844 expression inhibits NPC cell invasion and metastasis, at least in part, by suppressing PI3K–AKT signaling. Clinically, high ZNF844 expression correlates with improved OS and DMFS, highlighting its potential as both a prognostic biomarker and a therapeutic target. These findings provide new insights into the epigenetic regulation of NPC progression and suggest that strategies aimed at reactivating ZNF844 may offer therapeutic benefit.

Author Contributions

Jinping Xu: data curation, writing – original draft. Cancan Chen: validation, writing – original draft, writing – review and editing. Bin Zhang: formal analysis, resources, validation. Shufang Liao: data curation, investigation. Yunyan Mo: data curation, investigation. Rongjun Zhang: data curation, investigation. Xiangyun Kong: data curation, investigation. Wei Jiang: conceptualization, project administration, supervision.

Funding

This work was supported by grants from the Key Research and Development rogram of Guangxi [grant numbers AB24010078, AB25069454]; the Science Research and Technology Development Program of Guilin [grant number 20230127–1]; and the Guangxi Medical and Health Key Discipline Construction Project. This work was supported by the Guangxi Medical and Health Key Discipline Construction Project. The Science Research and Technology Development Program of Guilin, 20230127‐1, the Key Research and Development Program of Guangxi, AB24010078, AB25069454.

Ethics Statement

All human samples were obtained with informed consent from patients with nasopharyngeal carcinoma. Ethical consent was granted from the Ethical Committee Review Board of the Affiliated Hospital of Guilin Medical University (permit number: GLMC20240381). The animal study was reviewed and approved by the Ethical Committee Review Board of Guilin Medical University (permit number: GLMC20243385).

Consent

All authors have reviewed and approved the contents of this manuscript and unanimously consent to its publication.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Primers and siRNA sequences used in this study.

Table S2: Reverse transcription reaction system.

Table S3: Quantitative PCR reaction system.

Data Availability Statement

The datasets generated and analyzed in this study are available from the corresponding authors upon reasonable request via email.