NPC clinical sample collection

From 2014 to 2017, we procured 42 pairs of freshly frozen tumor tissues and adjacent normal nasopharyngeal epithelial tissues (situated at a distance greater than 3 cm from the tumor) from patients diagnosed with NPC undergoing treatment at our facility. Furthermore, 5 pairs of formalin-fixed, paraffin-embedded biopsy tissues from NPC and nearby tissues were collected. Prior to biopsy, all patients were histopathologically confirmed to have primary NPC and had not received any oncological treatments. Two NPC experts conducted histological verification of the collected tissues. The study adhered to the principles of the Declaration of Helsinki and received approval from the Ethics Committee of XuZhou Central Hospital. Informed consent was obtained from all subjects, and they were monitored for a follow-up period of five years.

Immunohistochemistry

The formalin-fixed, paraffin-embedded tissue sections underwent standard deparaffinization and rehydration, followed by antigen retrieval. Blocking of intrinsic peroxidase activity was achieved using 3% hydrogen peroxide at 25 degrees Celsius for 25 min. The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature. Imaging of representative areas was done using Aperio ImageScope software version 11.2.0.780 (Leica Biosystems, Wetzlar, Germany).

Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR).

Total ribonucleic acid (RNA) was extracted from the tissues and cells using TRIzol reagent (15,596,018, Thermo Fisher Scientific), then reverse transcribed to complementary DNA (cDNA) using PrimeScript RT master mix (RR058A, Takara, Japan). The RT-qPCR was performed using SYBR Green qPCR Super Mix-UDG (Thermo Fisher Scientific). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as the internal reference gene. Gene expression quantification was based on the 2−ΔΔCT method. Primer sequences used in the RT-qPCR are detailed in Table 1.

Table 1 RT-qPCR primer sequencesCell culture

Five NPC cell lines, namely HNE1, HNE2, CNE1, CNE2, and HONE1, were cultured in Roswell Park Memorial Institute-1640 medium (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, USA). The immortalized human nasopharyngeal epithelial cell line, NP69, was maintained in keratinocyte/serum-free medium (Invitrogen) supplemented with bovine pituitary extract (BD Bioscience, CA, USA). All cell lines, sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were incubated in a humidified atmosphere with 5% CO2 at 37 °C. The cell lines were authenticated using Short Tandem Repeat profiling by GENEWIZ Biotechnology Co., Ltd. (Suzhou, China) and routinely screened for mycoplasma contamination (Yeasen, # 40601ES20). To assess the impact of the compound FKC on NPC cell biological behavior, the cells were treated with 4 μM FKC for 48 h.

Cell transfection

The cDNA of HSP90B1 was amplified by PCR and subcloned into the pcDNA3.1 vector (Invitrogen), with the final construct confirmed by sequencing for overexpressing HSP90B1. The empty pcDNA3.1 vector was used as a control. Small interfering RNA (siRNA) targeting HSP90B1 (si-HSP90B1: 5′-atggattaaatgcatcacaaata-3′) and negative control (si-NC) were designed and provided by RiboBio (Guangzhou, China) for knocking down HSP90B1 and EGFR. When cell confluence reached 70%–80%, transfection with the aforementioned plasmids or oligonucleotides was performed using Lipofectamine 3000 (L3000015, Thermo Fisher Scientific) following the manufacturer's protocol. After 48 h, transfection efficiency was assessed by RT-qPCR and Western blot. To inhibit the EGFR signaling pathway, HNE1 and CNE2 cells were treated with cetuximab (Erbitux®, Merck KGaA, Darmstadt, Germany, 20 ng/ml).

Cell counting Kit-8 (CCK-8) assay

To evaluate the cell viability of NP69, cells (4500 cells/well) were seeded into a 96-well plate. Cells were treated with various concentrations of FKC (0.5, 1, 2, 4 μM) under 5% CO2 at 37 °C for 48 h. Each well was supplemented with 10 μL CCK-8 solution (Sangon Biotech, Shanghai, China), and incubated for an additional 4 h. The absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability (100%) = (OD of the experimental group—OD of the blank group)/(OD of the control group—OD of the blank group) × 100%.

For assessing cell proliferation, HNE1 and CNE2 cells were treated with different concentrations of FKC (0.5, 1, 2, 4 μM) for 48 h, then harvested and seeded into a 96-well plate (2000 cells/well). Cells were incubated in 96-well plates with 100 µl of complete medium with 5% CO2 at 37 °C. At designated time points (24, 48, and 72 h), 10 μL of CCK-8 solution (Sangon Biotech, Shanghai, China) was added to each well, followed by a 2 h incubation. Absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad) [23].

Colony formation assay

HNE1 and CNE2 cells were seeded in 6-well plates at a density of 500 cells per well and cultured for 2 weeks. After washing with Phosphate-Buffered Saline, the cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet (V5265, Sigma-Aldrich). The number of colonies (diameter ≥ 100 μm) was manually counted to reflect cell proliferative capacity.

5-Ethynyl-2'-deoxyuridine (EdU) Proliferation Assay.

Cell proliferation was determined using an EdU assay kit (C10310, RiboBio) according to the manufacturer's instructions. Briefly, treated cells were seeded into 6-well plates at a density of 5 × 103 cells/well and incubated with 100 μl of 50 μM EdU at 37 °C, followed by fixation with 4% paraformaldehyde. After nuclear staining with 4', 6-diamidino-2-phenylindole (DAPI), EdU-positive cells were observed and analyzed under a fluorescence microscope (Olympus, Japan) to reflect changes in cell proliferation. The EdU positive rate was calculated as EdU positive/DAPI × 100%.

Flow cytometry

For apoptosis assessment, HNE1 and CNE2, NPC cell lines, were harvested and stained using an Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (Vazyme) [24, 25]. Suspended and trypsinized cells were washed with ice-cold Dulbecco’s Modified Eagle Medium (DMEM) with Hank’s Balanced Salt Solution (D-Hanks solution). After centrifugation, cells were resuspended in binding buffer, mixed with 5 µL of Annexin V-FITC and 5 µL of propidium iodide, and incubated in the dark at room temperature for 15 min. Apoptotic rates were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Metabolic assay

The Extracellular Acidification Rate (ECAR) was determined using a Seahorse Biosciences XF96 Analyzer (North Billerica, MA, USA) [26]. HNE1 and CNE2 cells were seeded in XF96 well assay plates and incubated overnight. ECAR was measured under basal conditions and in response to 10 mM glucose, 5 μM oligomycin, and 100 mM 2-deoxyglucose (all from Sigma-Aldrich). The protocol included 3 min of mixing, 3 min of waiting, and 3 min of measurement, with ECAR values reported as milli-pH per minute (mpH/min).

Glucose consumption and lactate production assay

Glucose and lactate levels in the culture medium of HNE1 and CNE2 cells were quantified using the Glucose Assay Kit and Lactate Assay Kit (Abcam), respectively. Following the manufacturer's instructions, culture medium was incubated with reaction mixtures at room temperature for 30 min, and absorbance at 450 nm was measured using a Bio-Tek microplate reader.

Human umbilical vein endothelial cell (HUVEC) tube formation assay

Conditioned media from HNE1 and CNE2 cells were collected. Precooled Matrigel (BD Biosciences) was added to precooled 96-well plates (50 µL per well) and allowed to solidify at 37 °C for 30 min. HUVECs were seeded and incubated with conditioned media at 37 °C for 6 h. Tube formation was observed and captured using an inverted microscope (Olympus).

HUVEC migration assay

HUVECs were harvested and suspended in DMEM without FBS. A 400 µL cell suspension containing 10,000 cells was seeded into the upper chamber of a Millicell insert (Millipore, USA). NPC cell-conditioned medium (600 µL) was added to the lower chamber. After 36 h, cells in the Transwell chambers were fixed with 4% formalin (Sevicebio, Wuhan, China) and stained with 0.1% crystal violet for 30 min. Migrated cells were counted post washing with PBS and imaged using a microscope (Olympus) [27].

Co-immunoprecipitation (Co-IP) experiment

For Co-IP assays, approximately 107 HNE1 and CNE2 cells were lysed in IP buffer containing 50 mM Tris–HCl pH 7.4, 150 mM Sodium Chloride (NaCl), 2 mM Ethylenediaminetetraacetic Acid, and 1% Nonidet P-40, supplemented with 1 × protease inhibitor mixture and 1 mM Phenylmethylsulfonyl fluoride. Primary antibodies were added to the supernatants of the lysates and incubated for 2 h, followed by the addition of Protein A/G magnetic beads to each sample. The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Western blot

Cells and tissues were lysed in radioimmunoprecipitation assay buffer (Beyotime, Jiangsu, China) for protein extraction, and protein concentrations were determined using the bicinchoninic acid Protein Assay. Proteins were separated via SDS–Polyacrylamide Gel Electrophoresis (Beyotime) and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies against HSP90B1 (ab3674, Abcam), phosphorylated EGFR (p-EGFR, 3777, Cell Signaling Technology), EGFR (ab52894, Abcam), Glucose Transporter 1 (GLUT1, ab652, Abcam), Hexokinase 2 (HK2, 22,029-1-AP, Proteintech), Angiopoietin 1 (Ang-1, ab8451, Abcam), Vascular Endothelial Growth Factor (VEGF, ab46154, Abcam), PI3K (4249, Cell Signaling Technology), phosphorylated PI3K (p-PI3K, 4228, Cell Signaling Technology), Akt (4691, Cell Signaling Technology), phosphorylated Akt (p-Akt, 9271, Cell Signaling Technology), phosphorylated mTOR (p-mTOR, 5536, Cell Signaling Technology), mTOR (2972, Cell Signaling Technology), and Glyceraldehyde 3-Phosphate Dehydrogenase (2118, Cell Signaling Technology). Horseradish Peroxidase-conjugated secondary antibodies were used for further incubation, and immunoblots were visualized using Clarity Max Western ECL Substrate (32,134, Pierce Biotechnology, Rockford, IL, USA).

Tumor xenograft model in mice

This animal study was approved by the Animal Ethics Committee of XuZhou Central Hospital and conducted in strict accordance with the committee’s guidelines. Sixty male BALB/C nude mice, aged 5–6 weeks, were procured from the Charles River Laboratories (Hunan SJA Laboratory Animal Co., Ltd).

Mice were randomly divided into six groups: PBS group, FKC group, FKC + si-NC group, FKC + si-HSP90B1 group, FKC + pcDNA 3.1 group, and FKC + pcDNA 3.1-HSP90B1 group. The PBS and FKC groups were subcutaneously inoculated with HNE1 cells treated with PBS (2 × 106 cells/mouse), while the other groups received HNE1 cells with either stable knockdown or overexpression of HSP90B1 (2 × 106 cells/mouse). To determine the inhibitory effect of FKC on NPC tumor growth, treatment with FKC (3 mg/kg) was initiated via intraperitoneal injection when tumor volumes reached 70–120 mm3 [28]. The PBS group received an equivalent volume of vehicle solution (0.9% saline containing 4% Dimethyl sulfoxide and 5% Tween 80) as a control. Tumor volumes were measured weekly using calipers, with the volume calculated as length × width2/2. After 4 weeks, mice were euthanized, and tumor tissues were collected for gene analysis.

To assess the impact of FKC on tumor liver metastasis, HNE1 cells infected with a lentivirus expressing luciferase were used. Post-infection, cells were selected for efficient viral integration and stable luciferase expression using flow cytometry. Mice were intravenously injected with these cells (2 × 106 cells/mouse). Over the subsequent 8 weeks, specific FKC treatment (3 mg/kg) was administered. At the end of the experiment, intraperitoneal injection of luciferin solution (150 mg/kg) was performed for quantitative assessment of tumor metastasis using bioluminescence imaging [29].

Data analysis

Statistical analysis was conducted using SPSS software version 19.0, GraphPad Prism version 5, and ImageJ software. A two-tailed unpaired Student's t-test was used for comparisons between two groups. One-way Analysis of Variance was applied for multiple group comparisons. Pearson correlation analysis was performed to analyze the correlation between two genes. Survival curves were estimated using the Kaplan–Meier method and compared using the log-rank test. Data are presented as mean ± standard deviation (SD). A P-value of less than 0.05 was considered statistically significant.