The FaDu cell line, human head and neck squamous carcinoma cells [12] and sourced from the Korean Cell Bank, played a pivotal role in our investigative efforts. To cultivate these cells, a carefully curated environment was maintained, utilizing DMEM (Dulbecco’s modified eagle’s medium; Corning), enriched with 10% fetal bovine serum (FBS; Corning), and a 1% antibiotics solution consisting of penicillin and streptomycin (Thermo Fischer Scientific). This growth occurred under controlled conditions at 37 °C within a 95% humidified atmosphere infused with 5% CO₂.

Our research pursuits centered around achieving a nuanced and precise modulation of KCNK1 expression in FaDu cells. To attain this objective, we implemented targeted transfection strategies. Specifically, we introduced genetic constructs, including pEGFP-N1-hKCNK1, pCMV10-hKCNK1, scramble siRNA, and KCNK1-specific siRNA, into the cellular milieu, employing the lipofectamine 3000 reagent (Thermo Fischer Scientific). This sophisticated transfection approach was pivotal in granting us the capability to intricately control the expression levels of KCNK1.

Tissue pieces from HNSCC patient was obtained and subjected to incubation in dispase solution (GenDEPOT) at 37 °C for 1 hour. Following incubation, cells were isolated using a cell strainer with a pore size of 70 μm in conjunction with DMEM/F12 medium (Gibco) [13,14]. The cells were then centrifuged at 1,500 rpm for 3 minutes, after which the supernatant was discarded. The cell pellet was resuspended in Airway Epithelial Cell Growth Medium (PromoCell) supplemented with Growth Medium SupplementMix (PromoCell) and 1% antibiotic solution, consisting of penicillin and streptomycin (Thermo Fisher Scientific). To initiate primary cancer cell cultures, the resuspended cells were seeded in 6-well plates containing the growth medium.

RNA was isolated using the RNeasy Plus Mini kit (Qiagen) and complementary DNA (cDNA) was synthesized using a first-strand cDNA synthesis kit (Roche). Real-time quantitative polymerase chain reaction (PCR) was performed using SYBR Green Master (Roche Applied Science). cDNA was amplified by real-time PCR (LightCycler 480 Instrument II; Roche) according to the manufacturer’s recommendations. The primers used for amplifying PCR products were: 5´-ACT TCA CCT CCG CGC TCT TCT T-3´ (forward primer for hKCNK1), 5´-AAC AGG AGG GTG AAG GGA ATG C-3´ (reverse primer for hKCNK1), 5´-TTGAGGTCAATGAAGGGGTC-3´ (forward primer for GAPDH), and 5´-GAAGGTGAAGGTCGGAGTCA-3´ (reverse primer for GAPDH) [15]. Gene expression was quantified using the comparative threshold cycles of GAPDH as the reference gene.

In the in vitro assays for invasion or migration, 5×10⁴ FaDu cells were seeded into 24-well transwell inserts that had been either precoated with Matrigel (Corning) or left uncoated (SPL). In the transwell assay, the upper chambers were filled with 100 μL of medium without FBS, while the lower chambers of the transwell apparatus were filled with 750 μL of medium supplemented with 10% FBS [15,16]. Following a 72 hour incubation period, cells that had traversed the membrane were fixed with methanol and subsequently stained with hematoxylin and eosin solutions. For quantification purposes, at least five fields were randomly selected and captured using an inverted microscope, facilitating the counting of cells [15].

FaDu cells were cultured in 10 cm dishes and subjected to transfection with pGFP-N1-KCNK1 using Lipofectamine 3000 for a period of 24 hours. Post-transfection, the cells were assessed for viability through trypan blue exclusion staining and subsequently enumerated. A designated low density of viable cells, specifically 5×10³ cells per well, was plated onto six-well cell culture plates and allowed to proliferate for an additional 2 weeks. Following this period of growth, the cells underwent a double washing procedure with phosphate-buffered saline (PBS) and were then fixed using cold methanol for a duration of 15 minutes. After the fixation process, the cells were stained with methylene blue (prepared as a 1% solution in water) at ambient temperature for 15 minutes. The final step involved thoroughly rinsing the plates with distilled water to remove any excess stain, after which the plates were left to dry [17]. This methodical approach ensured a detailed analysis of cell morphology and viability post-transfection.

Zebrafish were maintained, handled, and bred according to standard protocols from the Institutional Animal Care Committee of Korea University. We kept adult zebrafish at a temperature between 26 °C and 28 °C, with lights on for 14 hours and off for 10 hours each day [18]. We got embryos by mating one male and two female zebrafish together in a tank. These embryos were then kept at 28.5 °C in E3 medium diluted with 0.2 mM phenylthiourea (PTU) for 48 hours to inhibit melanization [19]. Zebrafish wild type strain were obtained from Korea University Zebrafish Translational Medical Research Center.

Wild-type zebrafish larvae were cultured in E3 medium, which was supplemented with 0.2 mM PTU, until reaching 2 days post-fertilization (dpf) at a temperature of 28.5 °C. Subsequently, the embryos were dechorionated and anesthetized using a solution of 1× Tricaine (0.16 g/L Tricaine dissolved in E3 medium). For the purpose of transplantation, the anesthetized larvae were positioned on the lid of a petri dish, which had been previously coated with a 2% agarose solution, a method that has been outlined in prior studies. The injection of tumor cells was performed utilizing borosilicate glass capillaries (World Precision Instruments), which were fashioned into needles using a needle puller (Sutter Instruments). These needles, once loaded with approximately 10 μL of the tumor cell suspension, were affixed to a micromanipulator (World Precision Instruments) and connected to a microinjector (World Precision Instruments). Following this setup, the tumor cells were introduced into the yolk sac of the zebrafish larvae [19]. After the injection, the larvae were maintained at a temperature of 34 °C. At 1 day post-injection (dpi), transplanted larvae underwent a sorting process, which was based on the visual identification of tumor cells within the yolk sac [20]. When treatment with quinidine was required, zebrafish larvae were immediately transferred to an E3 medium with a concentration of 100 μM quinidine, which does not affect zebrafish mortality, and were raised for 2 days following the injection.

For the purpose of zebrafish injection, FaDu cells and patient-derived cells were stained with CM-DiI (Invitrogen) [21], initially incubated for 15 minutes at 37 °C followed by a subsequent 10-minute incubation at 4 °C. After labeling, cells were washed with PBS and subsequently resuspended in the appropriate growth medium. The cell suspension was adjusted to a final concentration of 1×10⁸ cells/mL. To evaluate cell viability post-staining and trypan blue staining was used to assess cell viability.

First, we took total RNA from FaDu cells treated with either KCNK1 siRNA or a scramble siRNA and extracted it using the RNeasy Plus Micro Kit. After cleaning this RNA, we prepared it for RNA sequencing by using the TruSeq Stranded mRNA Library Prep Kit. This process created cDNA libraries, which we then sequenced using HiSeq platforms by Illumina. We trimmed reads in the Illumina FASTQ format and mapped them to the human reference genome (GRCh38/hg19) using HISAT2 version 2.1.0. The StringTie program helped us assemble known genes/transcripts using a reference gene model. After assembling, we estimated the abundance of these transcripts by counting reads and using normalized values like FPKM (fragments per kilobase of transcript per million mapped reads) and TPM (transcripts per million). We used StringTie results to find genes that were significantly different between groups. We focused on genes that were either up- or down-regulated with a |log2 fold change|>2 and P-value <0.01 were selected. Based on these data, we conducted a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify pathways associated with KCNK1.

Data analysis was performed utilizing Prism software version 9.1 (GraphPad). The results are expressed as mean±standard deviation for measured variables. For quantitative data, statistical analysis was conducted using the Student t-test for two-group comparisons and one-way analysis of variance for comparisons among multiple groups, assuming normality of the data distribution. The chi-square test was applied for the analysis of categorical data. Pearson’s correlation analysis was used to assess the relationships between variables. A P-value of less than 0.05 was considered statistically significant.

To investigate the role of KCNK1 in the FaDu cell line, a type of head and neck cancer cell, we transfected these cells with pEGFP-N1-KCNK1 to overexpress KCNK1. Subsequently, we conducted a transwell migration assay. Compared to the control group of FaDu cells, the KCNK1-overexpressing group exhibited approximately twice the number of migrated cells (Fig. 1A and B). Additionally, we performed an invasion assay using a Matrigel-coated transwell. The results indicated that the KCNK1-overexpressing group had about three times more cells that invaded through the Matrigel (Fig. 1C and D).

Conversely, to determine the impact of KCNK1 knockdown on the migration and invasion of FaDu cells, we transfected these cells with KCNK1 siRNA and scramble siRNA. This approach established a group of FaDu cells with KCNK1 knockdown and a control group. We then performed transwell migration assays and invasion assays on these groups using the previously described methods (Fig. 2A and C). The findings revealed that the KCNK1 knockdown group exhibited a reduction in migration by approximately 50% and a decrease in invasion to less than half, compared to the control group (Fig. 2B and D).

Recent studies have shown that various K⁺ channels are critical in regulating cell proliferation. To investigate whether KCNK1 influences the proliferation of head and neck cancer cells, we performed a colony formation assay using FaDu cells with KCNK1 overexpression (Fig. 3A). The FaDu cells with KCNK1 overexpression formed approximately three times more colonies than the control group (Fig. 3B). These findings confirm that KCNK1 can modulate the proliferation capabilities of head and neck cancer cells.

To investigate the impact of KCNK1 overexpression in FaDu cells in vivo, we employed a zebrafish xenograft model. We transfected the FaDu cell line with either CMV10 or CMV10-KCNK1 to generate control FaDu cells and KCNK1-overexpressing FaDu cells, respectively. After performing live staining, we injected approximately 200 cells into the yolk sac of zebrafish larvae at 2 dpf and conducted live imaging using a confocal microscope at 1 and 3 dpi (Fig. 4A). The results showed that the tumor volume in the control group increased by about 2-fold, whereas the KCNK1-overexpressing FaDu cells exhibited a nearly three-fold increase in tumor volume (Fig. 4B). This suggests that tumors with KCNK1 overexpression grow more substantially. Additionally, we noted that the rate of micrometastasis, characterized by the spread of FaDu cells throughout the body of the zebrafish larvae, was significantly higher in the KCNK1 overexpression group at 64%, compared to 27% in the control group (Fig. 4C). This finding indicates that tumors from head and neck cancer cells overexpressing KCNK1 are more prone to metastasis.

Conversely, we sought to understand the effects of KCNK1 knockdown on head and neck cancer cell tumors. To achieve this, we utilized a zebrafish larvae xenograft model, introducing FaDu cells treated with KCNK1 siRNA to suppress KCNK1 expression. We then monitored the outcomes at 1 dpi and 3 dpi (Fig. 5A). The data showed that the control group experienced approximately a 30% increase in tumor growth from 1 dpi to 3 dpi, whereas the KCNK1 knockdown group exhibited negligible tumor growth (Fig. 5B). Furthermore, about 17% of the control group developed micrometastasis, in contrast to the KCNK1 knockdown group, where no micrometastasis was observed (Fig. 5C). These findings suggest that KCNK1 suppression can significantly reduce tumor growth and metastasis in head and neck cancer cells, highlighting KCNK1 as a promising therapeutic target for this type of cancer.

Quinidine, a Food and Drug Administration (FDA)-approved dpidrug known to inhibit various K⁺ channels, including KCNK1 [22,23], is used to restore normal sinus rhythm, treat atrial fibrillation and flutter, and manage ventricular arrhythmias [24]. Given the absence of a drug that selectively inhibits KCNK1, we explored the use of quinidine, which is effective among the currently FDA-approved drugs in inhibiting KCNK1, as an alternative. The objective was to assess the efficacy of quinidine in inhibiting growth and metastasis in head and neck cancer. For this purpose, we employed a zebrafish xenograft model developed with FaDu cells, treated with quinidine, and monitored the outcomes (Fig. 6A). The results showed that tumor growth in the quinidine-treated group was reduced by approximately 50% compared to the control group (Fig. 6B). Furthermore, the incidence of micrometastasis decreased from 54% in the control group to 27% in the quinidine-treated group, effectively halving the rate (Fig. 6C). These findings confirm that drugs targeting KCNK1 not only inhibit tumor growth in head and neck cancer cells but also significantly reduce metastasis.

To investigate the efficacy of the KCNK1 inhibitor quinidine on cells derived from patient head and neck cancer tumors, we conducted primary cell cultures using tissues from these tumors. We then stained the cells and established a zebrafish patient-derived xenograft (PDX) model. After administering quinidine to these cells and observing them within the yolk sac of the zebrafish at 1 dpi and 3 dpi using confocal microscopy (Fig. 7A), we observed that the tumor volume in the control group increased more than two-fold. In contrast, the quinidine-treated group showed only about a 10% increase in tumor volume (Fig. 7B). Although these results were not statistically significant, owing to the limited number of zebrafish PDX models used, a clear trend emerged indicating that quinidine inhibits tumor growth. Additionally, micrometastasis was observed in 50% of the control group, compared to 33% in the quinidine-treated group, suggesting a reduction in metastasis (Fig. 7C). Through this zebrafish xenograft PDX model using the KCNK1 inhibitor quinidine, we demonstrated the potential of KCNK1 as a therapeutic target for head and neck cancer.

To investigate the molecular mechanisms by which KCNK1 is involved in head and neck cancer cells, we knocked down KCNK1 in FaDu cells using KCNK1 siRNA. We then extracted RNA from these cells and from scramble siRNA control FaDu cells for RNA sequencing. This analysis identified a total of 72 differentially expressed genes (Fig. 8A and B). Through Gene Ontology and KEGG enrichment analysis, we examined pathways associated with KCNK1. The Gene Ontology analysis revealed that KCNK1 is related to various types of phospholipase activity (Fig. 8C). The phospholipase signaling network is well-known for its crucial roles in proliferation, migration, angiogenesis, and other processes in several cancers [25]. Additionally, the KEGG enrichment analysis indicated that KCNK1 is linked to numerous signaling pathways, including the Ras signaling pathway and the MAPK signaling pathway, which are known to be associated with tumor growth (Fig. 8D) [26,27]. Based on these findings, we propose that KCNK1 may regulate the Ras signaling pathway and the MAPK signaling pathway through its influence on the phospholipase signaling network, underscoring its pivotal role in the molecular dynamics of cancer tumor growth.