LUCAT1 Drives Arecoline-Induced Head and Neck Cancer Progression via STAT1-Mediated Transcriptional Regulation

LUCAT1 Drives Arecoline-Induced Head and Neck Cancer Progression via STAT1-Mediated Transcriptional Regulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article LUCAT1 Drives Arecoline-Induced Head and Neck Cancer Progression via STAT1-Mediated Transcriptional Regulation Hung-Han Huang, Guo-Rung You, Joseph T Chang, Ann-Joy Cheng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6980917/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Apr, 2026 Read the published version in Cellular & Molecular Biology Letters → Version 1 posted 15 You are reading this latest preprint version Abstract Background Areca nut chewing is a major environmental risk factor for head and neck cancer (HNC), particularly in Southeast Asia. However, the molecular mechanisms that link areca nut exposure to malignant progression remain poorly understood. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of oncogenesis, but their role in areca nut-associated HNC remains unexplored. Methods We performed functional assays, transcriptomic profiling, and bioinformatic analyses to investigate the role of the lncRNA LUCAT1 in arecoline-treated HNC cells. Cell motility, epithelial–mesenchymal transition (EMT), reactive oxygen species (ROS) levels, and therapeutic resistance were assessed following LUCAT1 knockdown or overexpression. We identified upstream regulators of LUCAT1 through promoter analysis, transcription factor knockdown, and pharmacological inhibition. Results LUCAT1 expression was significantly upregulated by arecoline exposure and promoted cell motility, EMT, ROS clearance, and resistance to radiotherapy and chemotherapy. Knockdown of LUCAT1 reversed these malignant phenotypes and suppressed antioxidant enzyme expression, partly through modulation of the p38 MAPK pathway. Transcriptomic and promoter analyses identified STAT1 as a key transcription factor activated by arecoline through muscarinic acetylcholine receptor (mAChR) signaling. Functional rescue experiments confirmed that LUCAT1 acts downstream of STAT1 to sustain arecoline-induced tumor aggressiveness. Conclusion Our findings define a novel mAChR–STAT1–LUCAT1 regulatory axis that mediates areca nut-induced malignant progression in HNC. This study not only reveals a critical molecular pathway linking environmental carcinogen exposure to oncogenic transcriptional reprogramming but also highlights LUCAT1 as a promising target for therapeutic intervention in high-risk HNC patients. Lung Cancer Associated Transcript 1 (LUCAT1) Signal Transducer And Activator Of Transcription 1 (STAT1) areca nut arecoline head and neck cancer Epithelial–mesenchymal transition (EMT) Reactive oxygen species (ROS) Therapeutic resistance p38 MAPK signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Head and neck cancer (HNC), encompassing malignancies of the lip, oral cavity, pharynx, and larynx, represents a significant global health challenge, with estimated 946,000 new cases and 482,000 deaths reported in 2022 [ 1 ]. Standard treatments, including surgery, radiotherapy, and chemotherapy, are effective for early-stage HNC, achieving five-year survival rates exceeding 80% [ 2 , 3 ]. However, advanced-stage HNC is characterized by tumor invasion, local recurrence, and resistance to therapies such as cisplatin, resulting in significantly reduced survival rates [ 2 – 4 ]. These clinical challenges highlight the need to elucidate the molecular mechanisms driving HNC progression and therapeutic resistance. HNC is associated with several etiological factors, including tobacco smoking, alcohol consumption, human papillomavirus (HPV) and Epstein–Barr virus (EBV) infections, and areca nut chewing [ 5 – 7 ]. Areca nut chewing, a culturally entrenched practice in Southeast Asia, is a major etiological factor due to its potent carcinogenic properties [ 7 , 8 ]. Arecoline, the main alkaloid in areca nut, induces genotoxic and cytotoxic effects in HNC cells through mechanisms such as DNA damage, oxidative stress, and epithelial–mesenchymal transition (EMT), all of which promote tumor progression [ 8 ]. Patients with a history of areca nut chewing often present with more aggressive tumor phenotypes, increased incidence of secondary primary tumors, reduced sensitivity to chemoradiotherapy, and poorer survival outcomes [ 9 , 10 ]. Despite these observations, the molecular regulators mediating areca nut-induced HNC phenotypes remain poorly understood. Long noncoding RNAs (lncRNAs), transcripts longer than 200 nucleotides with no protein-coding potential, have emerged as pivotal regulators in cancer biology [ 11 ]. By interacting with chromatin, RNA, or proteins, lncRNAs modulate transcriptional regulation, RNA stability, and signal transduction, influencing tumorigenesis processes such as proliferation, invasion, and cancer stemness [ 11 – 13 ]. Although areca nut is a well-established carcinogen in HNC, the role of lncRNAs in areca nut-induced carcinogenesis remains underexplored, with limited studies identifying dysregulated lncRNAs requiring further validation [ 14 , 15 ]. Our previous transcriptomic analysis identified 28 lncRNAs associated with areca nut-induced HNC, with LUCAT1 (Lung Cancer Associated Transcript 1) showing significant upregulation in response to arecoline treatment [ 15 ]. LUCAT1, also known as SCAL1, was initially reported to be elevated in the airway epithelia of cigarette smokers [ 16 ]. Subsequent studies have implicated LUCAT1 in multiple cancers, including lung, breast, liver, renal, and bladder cancers, where it promotes oncogenesis through mechanisms such as competitive binding to miRNAs (e.g., miR-4316/VEGF-A, miR-375/YAP1) or interactions with oncogenic proteins (e.g., IGF2BP2, HSP90) [ 17 – 24 ]. However, its role in areca nut-associated HNC remains uncharacterized. The present study investigates the mechanistic role of LUCAT1 in areca nut-induced HNC. We first delineate LUCAT1’s contribution to malignant phenotypes, including enhanced invasiveness and therapeutic resistance. We then demonstrate that arecoline upregulates Signal Transducer and Activator of Transcription 1 (STAT1), which transcriptionally activates LUCAT1. This arecoline–STAT1–LUCAT1 axis drives aggressive cancer phenotypes, including cell invasion, and cisplatin and radiation resistance. By elucidating this novel oncogenic pathway, our findings provide critical insights into areca nut-driven molecular carcinogenesis and identify LUCAT1 and STAT1 as potential targets for precision therapeutic strategies in areca nut-associated HNC. Methods Cell Culture and Arecoline Treatment Three HNC cell lines, OECM1 (RRID: CVCL_6782), CGHNC8, and CGHNC9, were used in this study [ 25 ]. OECM1 cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA), while CGHNC8 and CGHNC9 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific). Both media were supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific). Cells were incubated at 37°C in a humidified atmosphere with 5% CO₂. To model the chronic effects of areca nut exposure, arecoline-adapted sublines were generated by treating parental cell lines with arecoline (Sigma-Aldrich, St. Louis, MO, USA) at an IC₃₀ concentration in complete medium for three months, as previously described [ 15 ]. These sublines were used for subsequent cellular and molecular analyses. LUCAT1 and Related Gene Manipulation Stable knockdown of LUCAT1 was achieved using CRISPR-based AAVS1 knock-in technology. A short hairpin RNA (shRNA) targeting exon 5 of LUCAT1 (shLUCAT1) was cloned into an AAVS1 knock-in donor vector (GeneCopoeia, Rockville, MD, USA), and an AAVS1-specific single-guide RNA (sgRNA) was subcloned into the pCas-Guide vector (OriGene Technologies, Rockville, MD, USA). These constructs, along with a scramble shRNA control, were co-transfected into HNC cell lines (OECM1 and CGHNC8) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). GFP-positive cells were sorted using a FACSAria III Cell Sorter (BD Biosciences, San Jose, CA, USA) and expanded by limiting dilution in the presence of puromycin (1 µg/mL) to establish single-cell clones. Stable LUCAT1-knockdown clones were subsequently treated with arecoline at an IC30 concentration for three months, as described [ 15 ], to generate arecoline-adapted sublines. For LUCAT1 overexpression, the full-length LUCAT1 sequence (NCBI Reference Sequence: NR_103548.1) was cloned into the pcDNA3.1(−) vector (Thermo Fisher Scientific). The construct was transiently transfected into HNC cells using Lipofectamine 2000 for functional analyses. To investigate transcriptomic and transcription factor regulatory effects, small interfering RNAs (siRNAs) targeting LUCAT1, STAT1, CUX1, and IRF6 were transfected into HNC cells using Lipofectamine 2000, as previously described [ 15 ]. Transfections were performed according to the manufacturer’s protocol, with cells incubated at 37°C for 24 hours before downstream assays. Knockdown and overexpression efficiencies were validated by RT-qPCR. Oligonucleotide sequences are provided in Supplementary Table S1 . Migration, Invasion, and Adhesion Assays Cell migration was evaluated using an in vitro wound healing assay, as previously described [ 26 , 27 ]. HNC cells (4 × 10 4 ) were seeded in an ibidi culture-insert two-well system (ibidi, Gräfelfing, Germany). Once the cells attached, the inserts were removed and replaced with serum-free medium, and the gap area was photographed at regular intervals. Wound closure was quantified by measuring the gap area. Cell invasion was assessed using a Matrigel-coated transwell insert (Merck, Darmstadt, Germany) [ 26 , 27 ]. HNC cells (1.2×10 5 ) were seeded into the upper chamber of inserts pre-coated with 5% Matrigel (Corning, Corning, NY, USA). The lower chamber contained medium with 20% FBS as a chemoattractant. After 18–30 hours of incubation at 37°C, non-invading cells were removed from the upper surface. Invading cells on the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted under a microscope in five random fields per insert. Cell adhesion was measured using a solid-phase attachment assay. Ninety-six-well plates were pre-coated with 5% Matrigel and blocked with 0.5% bovine serum albumin (BSA) in serum-free medium. HNC cells (5×10 4 ) were seeded into each well and incubated at 37°C for 30 minutes. Non-adherent cells were removed by washing with 0.1% BSA-containing serum-free medium. Attached cells were fixed with 4% formaldehyde and stained with 0.1% crystal violet; the stain was dissolved in 10% acetic acid for absorbance measurement at 595 nm. Radiosensitivity and Chemosensitivity assay Radiosensitivity was determined using a clonogenic survival assay, as previously described [ 28 ]. HNC cells (OECM1 and CGHNC8) were seeded into 35-mm dishes, irradiated with 4 gray (Gy) using a Gammacell 3000 (MDS Nordion, Ottawa, ON, Canada), and incubated at 37°C for 7–10 days to allow colony formation. Colonies were fixed with 4% formaldehyde, stained with 0.1% crystal violet, and counted. Plating efficiency (PE) was calculated as the ratio of colonies formed to cells seeded in unirradiated controls. Survival fraction (SF) was determined by dividing the PE of irradiated cells by that of unirradiated controls. Chemosensitivity to cisplatin was evaluated using a Cell Counting Kit-8 (CCK-8) viability assay (Dojindo Laboratories, Mashiki-machi, Kumamoto, Japan), as previously described [ 27 ]. HNC cells (2,500 per well) were seeded into 96-well plates and allowed to adhere overnight at 37°C. Cells were treated with cisplatin (Sigma-Aldrich, St. Louis, MO, USA) at concentrations ranging from 0 to 5 µg/mL for 48 hours. CCK-8 reagent was added according to the manufacturer’s protocol, and absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated as the percentage of absorbance in treated cells relative to untreated controls. Measurement of Intracellular ROS Levels Intracellular ROS levels were measured using the 5-(6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) assay (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. HNC cells were incubated with 1–10 µM CM-H2DCFDA in PBS for 30 minutes at 37°C. Fluorescence intensity (excitation 488 nm, emission 525 nm) was measured using a Guava easyCyte Flow Cytometer (Cytek Biosciences, Fremont, CA, USA). Data analysis was performed using FlowJo software, with mean fluorescence intensity (MFI) used as the primary readout for ROS levels. Background fluorescence was determined using unstained cells. Results were expressed as normalization to the mode. Gene and Protein Expression Analysis Gene expression was quantified using reverse transcription quantitative polymerase chain reaction (RT-qPCR), as previously described [ 26 , 28 , 29 ]. Total RNA was extracted from HNC cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using a reverse transcription kit (Bionovas, Toronto, ON, Canada) with 1 µg of RNA. RT-qPCR was performed using SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) on a CFX96 Real-Time PCR Detection System (Bio-Rad). Primer sequences for target genes, including LUCAT1, CDH1, CDH2, GCLC, GCLM, GPX2, STAT1, and GAPDH (reference gene), are listed in Supplementary Table S1 . Relative gene expression was calculated using the 2⁻ ΔΔCt method, normalized to GAPDH. Protein expression was analyzed by Western blotting, as previously described [ 26 , 28 , 29 ]. HNC cells were lysed in CHAPS buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% CHAPS) and incubated on ice for 30 minutes. Lysates were centrifuged at 12,000 × g for 15 minutes at 4°C to collect the supernatant. Protein was quantified via the Bradford assay (Bio-Rad, Hercules, CA, USA) followed by mixing with sample buffer and heating at 95℃ for 5 minutes. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Merck, Darmstadt, Germany). Membranes were blocked with 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween 20) and incubated overnight at 4°C with primary antibodies against target proteins, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Antibody details are provided in Supplementary Table S1 . Protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Merck) and imaged with an Amersham™ Imager 600 (GE Healthcare). Band intensities were quantified using ImageJ software and normalized to GAPDH. RNA sequencing Transcriptomic profiling was performed using RNA sequencing (RNA-seq) to compare differential gene expression between parental and LUCAT1 stable knockdown HNC cell lines (CGHNC9 and OECM1) and to elucidate LUCAT1’s downstream regulatory profile. Total RNA was extracted from HNC cells using TRIzol reagent according to the manufacturer’s protocol, and RNA integrity was assessed using the Caliper Labchip GX System (PerkinElmer, Waltham, MA, USA). Strand-specific RNA libraries were prepared with the TruSeq Stranded mRNA Sample Preparation kit (Illumina, San Diego, CA, USA), and library quality was verified using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Sequencing was conducted on a NextSeq 500 (Illumina, San Diego, CA, USA) with 150-bp paired-end reads. Raw RNA-seq data quality was confirmed using FastQC, and alignment was performed using the GDC mRNA analysis pipeline against GENCODE v43 (GRCh38.p13). Gene expression was quantified as TPM via the RSEM package (v1.3.3). Chromatin immunoprecipitation followed by RT-qPCR (ChIP-qPCR) Chromatin immunoprecipitation followed by RT-qPCR was based on the protocol provided by Abcam. Briefly, cells were fixed and cross-linked with formaldehyde and quenched with glycine. Nuclear fraction isolation was enriched by nuclear extraction buffer, and the chromatin was sheared by sonication to generate 150–300 bp fragments. Following sonication, chromatin was incubated with anti-STAT1 or species-matched normal IgG antibody as a negative control, pre-coated on Qbeads-protein G (MagQu Co., Ltd., New Taipei City, Taiwan) overnight at 4℃. The targets were enriched and pulled down by antibody and detected by RT-qPCR. Data were analyzed using fold enrichment compared to IgG controls. Bioinformatics Analysis Bioinformatic analyses were performed on RNA sequencing (RNA-seq) data from parental and LUCAT1 stable knockdown HNC cell lines (CGHNC9 and OECM1) to investigate LUCAT1-mediated biological pathways and transcriptional regulation. Gene Set Variation Analysis (GSVA) was conducted using the GSVA R package (v1.50.5) to assess pathway activity across the transcriptomic dataset [ 30 ]. Gene Set Enrichment Analysis (GSEA) was performed using GSEA software (v4.2.3) to identify concordant differences in gene expression patterns between experimental groups. To investigate LUCAT1 transcriptional regulation, the LUCAT1 promoter region was defined as spanning − 2000 to + 100 base pairs relative to the transcription start site (TSS). DNase I hypersensitivity sites within this region were identified using ENCODE data accessed via the UCSC Genome Browser (GRCh38) to detect potential active regulatory elements. Transcription factor (TF) binding motifs were predicted using the JASPAR 2022 database, applying a position weight matrix (PWM) score threshold > 0.8 and p < 0.05. TFs with predicted binding sites overlapping DNase I hypersensitivity regions were prioritized as candidate regulators of LUCAT1 transcription. Data Processing and Statistical Analysis All experiments were conducted in at least triplicate unless otherwise specified. Data are reported as mean ± standard deviation (SD). Statistical comparisons between two groups were performed using two-tailed Student’s t-tests. For comparisons involving multiple groups or conditions, one-way or two-way analysis of variance (ANOVA) was used, followed by post-hoc Tukey’s tests for pairwise comparisons. Statistical analyses were performed using R (v4.3.3) and GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant, unless otherwise indicated (e.g., FDR for GSEA). Results LUCAT1 Silencing Abrogates Cell Motility in HNC cells by inhibiting EMT To elucidate the role of LUCAT1 in HNC progression, stable LUCAT1 knockdown cell lines (sh-LUCAT1) were generated in OECM1 and CGHNC8 cell lines using CRISPR-based AAVS1 knock-in technology. Cell motility phenotypes, including invasion, migration, and adhesion, were assessed using Matrigel invasion assays, wound-healing assays, and solid-phase attachment assays, respectively. LUCAT1 depletion significantly reduced cell invasion in OECM1 and CGHNC8 cells compared to scramble controls (Fig. 1 A). Similarly, migration was inhibited at 12 hours (Fig. 1 B). In contrast, cell adhesion was enhanced following LUCAT1 overexpression (Fig. 1 C), suggesting that LUCAT1 modulates cell-extracellular matrix (ECM) interactions. Given the critical role of EMT in cancer cell motility and invasion [ 31 ], EMT marker expression was examined. RT-qPCR analysis revealed increased CDH1 (E-cadherin) and decreased CDH2 (N-cadherin) mRNA levels in LUCAT1-knockdown cells (Fig. 1 D). Western blotting confirmed these changes at the protein level, showing higher E-cadherin and lower N-cadherin expression in LUCAT1-silencing cells (Fig. 1 E). These findings indicate that LUCAT1 promotes HNC cell motility by inducing EMT, and its silencing inhibits invasive phenotypes through EMT suppression. LUCAT1 Silencing Enhances Radio- and Chemosensitivity in HNC Cells via Modulation of ROS Homeostasis To investigate the impact of LUCAT1 on therapeutic resistance, we assessed radiosensitivity and chemosensitivity in LUCAT1-knockdown OECM1 and CGHNC8. A clonogenic survival assay following 4 Gy irradiation revealed a reduction in surviving colonies compared to controls, indicating enhanced radiosensitivity (Fig. 2 A). Chemosensitivity to cisplatin was evaluated and presented increased cisplatin sensitivity, reducing cell viability in LUCAT1-knockdown cell lines (Fig. 2 B). Given that radiotherapy and chemotherapy induce DNA damage in part via reactive oxygen species (ROS) generation [ 32 ], we investigated LUCAT1’s impact on ROS levels. HNC cells overexpressing LUCAT1 (via pcDNA3.1-LUCAT1) were treated with camptothecin (CPT), and ROS production was measured using the H 2 DCFDA assay. LUCAT1 overexpression significantly reduced ROS accumulation compared to vector controls following CPT treatment (Fig. 2 C). To elucidate the mechanism, we analyzed ROS-scavenging enzymes (GCLC, GCLM, and GPX2) in LUCAT1-knockdown cells. RT-qPCR showed reduced mRNA levels of GCLC , GCLM , and GPX2 (Fig. 2 D), with Western blotting confirming decreased protein expression (Fig. 2 E). These results suggest that LUCAT1 contributes to therapeutic resistance in HNC cells by upregulating ROS-scavenging enzymes, and its silencing enhances sensitivity by disrupting ROS homeostasis. LUCAT1 Knockdown Reverses Arecoline-Induced Invasive and Resistance Phenotypes in HNC Cells Our prior studies identified LUCAT1 as an arecoline-inducible lncRNA in HNC cells [ 26 ]. To determine whether LUCAT1 mediated arecoline-driven malignant phenotypes, we chronically treated stable LUCAT1-knockdown (sh-LUCAT1) and control OECM1 and CGHNC8 cells with arecoline (IC30 dose-adaption) and assessed cell motility and therapeutic sensitivity. Arecoline treatment increased cell invasion in control cells, but this effect was significantly attenuated in LUCAT1-knockdown cells (Fig. 3 A). Similarly, arecoline-enhanced migration was reduced to near baseline levels in LUCAT1-knockdown cells at 12 hours (Fig. 3 B). For therapeutic sensitivity, arecoline increased radioresistance in control cells, with higher surviving colonies post-4 Gy irradiation, but LUCAT1 knockdown abrogated this effect (Fig. 3 C). Likewise, arecoline-induced cisplatin resistance was reversed, with LUCAT1-knockdown cells showing significantly lower viability (Fig. 3 D). To elucidate the molecular mechanisms underlying these phenotypes, we analyzed EMT markers and ROS-scavenging enzymes. Arecoline significantly decreased CDH1 (E-cadherin) mRNA and increased CDH2 (N-cadherin) mRNA in control cells, indicative of EMT induction, but these changes were reversed in LUCAT1-knockdown cells (Fig. 4 A). Western blotting confirmed restored E-cadherin and reduced N-cadherin protein levels in LUCAT1-knockdown cells (Fig. 4 B). For therapeutic sensitivity, arecoline upregulated ROS-scavenging enzyme GCLC , GCLM , and GPX2 mRNA in control cells, but these increases were significantly suppressed in LUCAT1-knockdown cells (Fig. 4 C), with similar reductions in proteins (Fig. 4 D). These findings demonstrate that LUCAT1 is critical for arecoline-induced malignant phenotypes in HNC cells. Specifically, LUCAT1 facilitates arecoline-driven cell motility by promoting EMT, as evidenced by altered E-cadherin and N-cadherin expression, and enhances therapeutic resistance by upregulating ROS-scavenging enzymes, thereby reducing intracellular chemotherapy-induced ROS levels. Silencing LUCAT1 effectively reverses these effects, underscoring its pivotal role in areca nut-associated HNC progression. LUCAT1 Facilitates EMT and Modulates ROS Clearance via p38 MAPK Signaling in Arecoline-Treated-HNC Cells To elucidate LUCAT1’s downstream regulatory network in HNC, we performed RNA sequencing (RNA-seq) on control and LUCAT1-knockdown (si-LUCAT1) OECM1 and CGHNC9 cell lines. Genes with a |fold change| ≥ 1.5 were identified, revealing 290 commonly upregulated and 503 commonly downregulated genes across both cell lines upon LUCAT1 silencing (Fig. 5 A, Supplementary Table S2 ). Notably, CGHNC9 cells, derived from an areca nut chewer [ 25 ], exhibited more pronounced transcriptional changes than OECM1 cells, suggesting LUCAT1’s role in areca nut-associated gene dysregulation. Gene set variation analysis (GSVA) was conducted using the GSVA R package to assess pathway enrichment in LUCAT1-knockdown cells. Despite differences in baseline transcriptomes, both cell lines showed consistent enrichment patterns, including oncogenic signaling (e.g., EGFR tyrosine kinase inhibitor resistance, VEGF signaling), cell motility (e.g., ECM-receptor interaction), and stress response (e.g., DNA damage repair, p53 signaling, glutathione metabolism) (Figs. 5 B, C). These pathways aligned with our functional findings of enhanced motility, EMT, and therapeutic resistance (Figs. 1 – 4 ). To evaluate LUCAT1’s role in areca nut-induced transcriptional reprogramming, we perform gene set enrichment analysis (GSEA) using GSEA software, comparing the shared LUCAT1-regulated gene set (up- and downregulated genes) with a published areca nut-induced downregulated gene set [ 30 ]. A significant negative correlation was observed (Fig. 5 D, Supplementary Table S3 ), indicating that LUCAT1 orchestrates areca nut-driven gene expression changes in HNC cells. The enrichment of glutathione metabolism, consistent with LUCAT1’s regulation of ROS-scavenging enzymes (Figs. 2 D–E, 4 C–D), prompted further investigation into oxidative stress mechanisms. Given that p38 MAPK signaling enhanced antioxidant enzyme expression to reduce intracellular ROS levels [ 33 , 34 ], we hypothesized that LUCAT1 modulated ROS via p38 MAPK activation. Arecoline treatment markedly increased phosphorylated p38 (p-p38) levels across both cell lines, while LUCAT1 knockdown significantly attenuated p-p38 levels with minimal effect on total p38 levels (Fig. 5 E). To validate p38’s role, we treated cells with anisomycin, a p38 activator [ 35 ]. Anisomycin increased p-p38, upregulated GPX2, and suppressed E-cadherin, mimicking arecoline’s effects, but these changes were significantly attenuated in LUCAT1-knockdown cells (Fig. 5 F). These results demonstrate that LUCAT1 acts as an upstream regulator of p38 MAPK signaling in arecoline-treated HNC cells, promoting EMT and modulating ROS through enhanced expression of ROS-scavenging enzymes, thereby driving cancer progression via increased motility and oxidative stress tolerance. Identification of STAT1 and CUX1 as Key Transcription Factors Regulating LUCAT1 Expression in Arecoline-Treated HNC Cells To investigate the transcriptional regulation of LUCAT1 in response to arecoline, we analyzed the LUCAT1 promoter region defined as the genomic interval spanning − 2000 to + 100 base pairs relative to the transcription start site, using the UCSC Genome Browser. Transcription factor (TF) binding sites were predicted using two complementary bioinformatics platforms: JASPAR 2022 [ 36 ] and ENCODE [ 37 ]. JASPAR provided curated position weight matrices (PWMs) to identify high-confidence TF binding sites (PWM > 0.8, p < 0.05), while ENCODE’s DNase I hypersensitivity site (DHS) data highlighted active regulatory regions. Three DHS-rich regions were identified within the LUCAT1 promoter: chr5:91314302–91314570, chr5:91315480–91315630, and chr5:91315800–91315950 (Fig. 6 A). JASPAR analysis predicted 494 candidate TFs potentially binding these DHS-defined loci (Supplementary Table S4 ). To prioritize TFs relevant to arecoline-induced LUCAT1 expression, we integrated the predicted TFs with a published areca nut-induced proteomic dataset identifying 1,083 differentially expressed proteins in areca nut-adapted HNC cell sublines [ 29 ]. Cross-referencing revealed three overlapped TFs—STAT1, IRF6, and CUX1—as potential regulators of LUCAT1 transcription (Fig. 6 B). To validate their regulation of LUCAT1, we performed siRNA-mediated knockdown of STAT1, IRF6, and CUX1 in OECM1 and CGHNC8 cells, with or without arecoline treatment (IC30 dose-adaption) and assessed LUCAT1 expression by RT-qPCR. Western blotting confirmed TF knockdown efficiency (Fig. 6 C). Silencing STAT1 or CUX1 significantly decreased LUCAT1 expression and attenuated arecoline-induced LUCAT1 levels (Fig. 6 D). In contrast, IRF6 knockdown modestly reduced LUCAT1 levels in arecoline-treated cells but increased LUCAT1 expression in control cells, suggesting a limited, context-dependent role (Fig. 6 D). These findings indicate that STAT1 and CUX1 are critical transcriptional activators of LUCAT1 in arecoline-treated HNC cells, with IRF6 exerting a minor regulatory influence. STAT1 Activation Induced LUCAT1 Expression in Response to Arecoline Exposure Prior studies have established that STAT1 serves as a transcription factor regulating oncogenic pathways in HNC [ 38 ], prompting further investigation of STAT1’s role in LUCAT1 expression. To confirm STAT1’s regulatory function, we treated OECM1 and CGHNC8 cells with fludarabine, a STAT1 inhibitor [ 39 ], at 0–30 µM. Western blotting showed a dose-dependent reduction in total STAT1 and phosphorylated STAT1 (p-STAT1) levels (Fig. 7 A). Correspondingly, RT-qPCR revealed a dose-dependent decrease in LUCAT1 expression (Fig. 7 B), supporting STAT1’s role as a transcriptional activator of LUCAT1. We hypothesized that arecoline enhances STAT1 activation, thereby upregulating LUCAT1 expression. We validated that arecoline treatment (IC30 dose-adaption) increases total and p-STAT1 in both cell lines (Fig. 7 C). To confirm if STAT1 regulates LUCAT1 expression via its promoter, we performed a ChIP assay. The result revealed that STAT1 indeed interacts with LUCAT1 promoter regions, especially DHS (Fig. 7 D). Given that arecoline is a known muscarinic acetylcholine receptor (mAChR) agonist [ 40 ], we tested whether STAT1 activation is mAChR-dependent using atropine, an mAChR antagonist [ 41 ]. Atropine (200 µM) reduced total STAT1 and p-STAT1 levels, decreased both phosphorylated and total STAT1 levels, and significantly attenuated arecoline-induced STAT1 activation (Fig. 7 E). In parallel, LUCAT1 expression was significantly decreased with atropine treatment in both parental and arecoline-adapted cells (Fig. 7 F). These results demonstrate that arecoline induces LUCAT1 expression through mAChR-mediated STAT1 activation, establishing a mechanistic link between areca nut exposure and oncogenic LUCAT1 upregulation in HNC cells. The STAT1-LUCAT1 Axis Drives Arecoline-Induced Invasive and Resistance Phenotypes in HNC To confirm the functional interplay between STAT1 and LUCAT1, we investigated whether LUCAT1 overexpression could rescue the phenotypic effects of STAT1 silencing in OECM1 and CGHNC8 cells treated with arecoline (IC30 dose-adaptation). LUCAT1 overexpression increased invasion, while STAT1 silencing reduced invasion compared to control cells (Fig. 8 A). Importantly, ectopic LUCAT1 expression significantly restored the invasion ability in STAT1-silenced cells, indicating that LUCAT1 acts downstream of STAT1 in regulating invasiveness. Similarly, LUCAT1 overexpression enhanced migration, whereas STAT1 silencing decreased migration; LUCAT1 overexpression reversed this migration defect in STAT1-silenced cells (Fig. 8 B). For radiosensitivity, LUCAT1 overexpression conferred resistance, increasing colony survival, while STAT1 silencing increased sensitivity by reducing survival colonies compared to control levels (Fig. 8 C). Crucially, LUCAT1 overexpression counteracted the radiosensitizing effect of STAT1 silencing, restoring colony survival to near control levels. These findings demonstrate that LUCAT1 functions as a critical downstream effector of STAT1 in arecoline-treated HNC cells, mediating STAT1-driven invasive and resistant phenotypes. The STAT1-LUCAT1 regulatory axis is a central mechanism by which areca nut exposure promotes HNC progression, orchestrating enhanced cell motility and therapeutic resistance through arecoline-induced transcriptional activation. Discussion The escalating global burden of HNC, particularly in regions where areca nut chewing is prevalent, underscores the urgent need to elucidate the molecular mechanisms driving its progression [ 7 , 8 ]. In this study, we identified the lncRNA LUCAT1 as a critical downstream effector of areca nut-induced oncogenic signaling in HNC. We demonstrated that LUCAT1 promotes cell motility through the induction of EMT and enhances radio- and chemoresistance by modulating ROS homeostasis (Figs. 1 , 2 ). Knockdown of LUCAT1 reversed arecoline-induced invasive and resistant phenotypes, concomitant with attenuation of EMT markers and suppression of ROS-scavenging enzyme expression (Figs. 3 , 4 ), partially mediated through the p38 MAPK signaling pathway (Fig. 5 ). Mechanistically, we discovered that LUCAT1 expression is transcriptionally regulated by STAT1, which is activated in response to arecoline stimulation (Figs. 6 , 7 ). Furthermore, we established that arecoline-mediated STAT1 activation occurs via mAChR signaling (Fig. 7 ), thereby linking environmental carcinogen exposure to a defined transcriptional regulatory network. Functional rescue experiments confirmed that LUCAT1 acts downstream of STAT1 to drive arecoline-induced invasion, migration, and radioresistance in HNC cells (Fig. 8 ). Collectively, these findings establish LUCAT1 as a pivotal lncRNA in arecoline-induced HNC progression, with the newly identified mAChR–STAT1–LUCAT1 signaling axis representing a promising molecular target for therapeutic intervention (Fig. 9 ). LUCAT1 has been implicated in multiple cancers [ 17 – 24 ]. Previous studies identified LUCAT1 as a driver of proliferation and metastasis in lung and breast cancers, in part through EMT induction [ 17 – 20 ]. Consistent with these reports, we demonstrate that LUCAT1 promotes cell motility and EMT in HNC, while its silencing suppresses invasion and migration, thus extending its oncogenic role to an arecoline-driven context. LUCAT1 has also been shown to promote chemoresistance and cancer stemness in lung, breast, and bladder cancers [ 21 – 23 ]. In line with these findings, we reveal that LUCAT1 enhances arecoline-induced radio- and chemoresistance in HNC and further demonstrate that LUCAT1 upregulates antioxidant enzymes such as GCLC, GCLM, and GPX2, thereby enhancing therapeutic resistance. These results establish a comprehensive mechanism by which areca nut promotes malignancy through LUCAT1 upregulation. Our transcriptomic profiling further supported these observations, showing that the LUCAT1-associated mRNA signature closely aligns with the transcriptional changes induced by arecoline (Fig. 5 D). Functional pathway analysis revealed enrichment of stress-response pathways, including JAK–STAT, cAMP signaling pathways, and glutathione metabolism (Fig. 5 B, C). Previous studies reported that p38 MAPK activation under cellular stress conditions can induce antioxidant genes and facilitate EMT, invasion, and therapy resistance [ 33 , 42 – 44 ], and that chronic areca nut exposure can activate the p38 pathway [ 45 , 46 ]. Our results are consistent with these findings, demonstrating that LUCAT1 promotes EMT and ROS modulation through p38 MAPK signaling (Fig. 5 F) and uniquely linking p38 regulation to LUCAT1 in arecoline-treated HNC cells (Fig. 5 E). Thus, LUCAT1 functions upstream of p38 MAPK to promote cell motility and resistance to therapy via EMT induction and ROS clearance. While arecoline has been previously associated with DNA damage, oxidative stress, and EMT induction in HNC cells [ 8 , 10 , 47 ], the specific transcriptional mediators of these carcinogenic effects were previously unknown. Here, we identify LUCAT1 as an arecoline-inducible lncRNA and further elucidate its upstream transcriptional regulators. Through integrated bioinformatic and experimental approaches, we found that STAT1, CUX1, and IRF6 potentially bind to the LUCAT1 promoter (Figs. 6 A, B) and are upregulated by arecoline exposure (Fig. 6 C). Among these, STAT1 and CUX1 were validated as key transcriptional activators of LUCAT1, whereas IRF6, despite being induced by arecoline and implicated in inflammation-related lncRNA-mRNA networks [ 14 ], did not significantly regulate LUCAT1 expression in our system (Figs. 6 C, D). The enrichment of the JAK–STAT signaling pathway among LUCAT1-regulated genes (Fig. 5 C) further pointed to STAT1 as a critical node warranting deeper investigation. STAT1 is classically known as a mediator of interferon signaling, capable of exerting both tumor-promoting and tumor-suppressive roles depending on the cellular context [ 48 ]. Recent studies, however, have demonstrated that STAT1 acts as an oncogenic driver in HNC, promoting cell proliferation, metastasis, and radioresistance [ 49 , 50 ]. Consistent with these observations, our data show that arecoline exposure upregulates STAT1 expression and phosphorylation (Fig. 7 C), leading to the activation of LUCAT1-mediated malignant phenotypes (Fig. 8 ). These results underscore the pivotal role of STAT1 as an upstream regulator of LUCAT1 under arecoline stimulation. Furthermore, we demonstrated that STAT1 activation by arecoline is dependent on muscarinic acetylcholine receptor (mAChR) signaling, as treatment with atropine—an mAChR antagonist—attenuated both total and phosphorylated STAT1 levels (Fig. 7 D) and concomitantly reduced LUCAT1 expression (Fig. 7 E). Together, these findings establish a novel mechanistic link wherein arecoline activates the STAT1–LUCAT1 axis via mAChR signaling, driving oncogenic transcriptional reprogramming and malignant progression in HNC. Conclusions This study uncovers a novel mAChR–STAT1–LUCAT1 signaling axis that links arecoline exposure to transcriptional reprogramming, EMT induction, ROS scavenging, facilitates cell motility and therapy resistance in head and neck cancer. By demonstrating that LUCAT1 functions as a central downstream effector of STAT1-mediated oncogenic signaling, our findings provide a mechanistic framework that explains how an environmental carcinogen drives malignant transformation at the molecular level. These results not only expand the functional understanding of LUCAT1 and STAT1 in HNC but also establish this axis as a promising target for therapeutic intervention in areca nut-associated cancer. Abbreviations HNC: head and neck cancer; HPV: human papillomavirus; EBV: Epstein–Barr virus; EMT: epithelial–mesenchymal transition; ROS: reactive oxygen species; LncRNA: long noncoding RNA; LUCAT1: lung cancer-associated transcript 1; HSP90: heat shock protein 90; STAT: signal transducer and activator of transcription; YAP1: Yes1 Associated Transcriptional Regulator; MAPK: Mitogen-activated protein kinase; FBS: Fetal Bovine Serum; AAVS1: adeno-associated virus integration site 1; IC30: 30% maximal inhibitory concentration; CUX1: Cut Like Homeobox 1; IRF6: Interferon regulatory factor 6; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TPM: transcripts Per Million; RSEM: RNA-Seq by Expectation-Maximization; GSVA: gene Set Variation Analysis; GSEA: gene set enrichment analysis; PWM: Position weight matrix; ECM: extracellular matrix; CPT: camptothecin; GCLC: Glutamate–cysteine ligase catalytic subunit ; GCLM: Glutamate–Cysteine Ligase Modifier Subunit; GPX2: Glutathione Peroxidase 2; Pt: parental subline; Arc: arecoline subline; JAK: Janus kinase; TF: transcription factor; mAChR: muscarinic acetylcholine receptor; ATF2: Activating transcription factor 2 Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The authors confirm that every dataset generated or analyzed during this study is either presented within this published article or can be obtained from the corresponding authors upon reasonable request. Competing interests Not applicable Funding This research was supported by grants from the Chang Gung Memorial Hospital–Linkou Medical Center (CMRPD1N0361). Authors' contributions Hung-Han Huang: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Software, Validation, Visualization, Writing–original draft. Guo-Rung You: Conceptualization, Methodology, Software, Writing–review & editing. Joseph T. Chang: Funding acquisition, Project administration, Resources, Supervision, Writing–review & editing. Ann-Joy Cheng: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing–review & editing. Acknowledgements The RNA-sequence and bioinformatics analyses were performed by Molecular Medicine Research Center and Bioinformatics Core Laboratory, Chang Gung University, Taiwan. References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. 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Supplementary Files SupplementaryTable1.docx SupplementaryTable2.xlsx SupplementaryTable3.xlsx SupplementaryTable4.xlsx SupplementaryuncroppedGelsandBlotsimage.pdf Cite Share Download PDF Status: Published Journal Publication published 22 Apr, 2026 Read the published version in Cellular & Molecular Biology Letters → Version 1 posted Editorial decision: Revision requested 07 Oct, 2025 Reviews received at journal 02 Oct, 2025 Reviews received at journal 26 Sep, 2025 Reviews received at journal 23 Sep, 2025 Reviews received at journal 16 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers agreed at journal 06 Sep, 2025 Reviewers agreed at journal 04 Sep, 2025 Reviewers agreed at journal 04 Sep, 2025 Reviewers agreed at journal 04 Sep, 2025 Reviewers agreed at journal 04 Sep, 2025 Reviewers invited by journal 13 Aug, 2025 Submission checks completed at journal 11 Aug, 2025 First submitted to journal 06 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6980917","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502082906,"identity":"5d3677b3-eef5-418e-98d5-d2faaa618a80","order_by":0,"name":"Hung-Han Huang","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Hung-Han","middleName":"","lastName":"Huang","suffix":""},{"id":502082907,"identity":"2c42ea7e-da8a-4969-8589-98a77b4a3557","order_by":1,"name":"Guo-Rung You","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Guo-Rung","middleName":"","lastName":"You","suffix":""},{"id":502082908,"identity":"6e264357-6fb4-4cb7-b232-5cb63adfac3b","order_by":2,"name":"Joseph T Chang","email":"","orcid":"","institution":"Linkou Chang Gung Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"T","lastName":"Chang","suffix":""},{"id":502082909,"identity":"399c0a0c-f1ce-4e08-a04f-79c9e19a34c5","order_by":3,"name":"Ann-Joy Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABHElEQVRIiWNgGAWjYJACAyBOAGLGBwgxNuK0MBugaOEhYBNIC5sEUVr4Z/ceKPjYZpfHL91+rfJLzR0G/v41Bgwfyg4z2EskYNUicedcguHMtuRiyTlnym7LHHvGIHHjjQHjjHOHGXhwaGG4kWNgzNt2IHHDjZy02xJshxkMJM4YMPO2AbVIY9ciD9OyH6ilWOIfVMtfPFoM4LZIpB9j/AhUacDfY8DMiEeLIVCL4YxzyYkzbuQwSzP2PeORuMFWcLDnXDoPz/0HWLXI3cgxM/hQZpfYPyP94ccf3+7I8fcf3vjgR5m1HHvPAezeB0aBASM4rnkMmHkYDvAwAAMKpBZfTDI/YPgDotkfMP4AKebHafgoGAWjYBSMUAAAfrFkTMe7lSkAAAAASUVORK5CYII=","orcid":"","institution":"Chang Gung University","correspondingAuthor":true,"prefix":"","firstName":"Ann-Joy","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2025-06-26 07:53:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6980917/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6980917/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s11658-026-00925-9","type":"published","date":"2026-04-22T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89562332,"identity":"68ab725f-ae7c-4464-8c98-d3245c9ee52a","added_by":"auto","created_at":"2025-08-21 10:24:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4397597,"visible":true,"origin":"","legend":"\u003cp\u003eLUCAT1 silencing suppresses cell motility in head and neck cancer (HNC) cells. (A) Matrigel invasion assay showing reduced invasive capacity of LUCAT1-stable knockdown (sh-LUCAT1) cells compared to controls in OECM1 and CGHNC8 cell lines. (B) Wound-healing assay demonstrating decreased migration ability in sh-LUCAT1 cells at 12 hours post-gap formation. (C) Solid-phase adhesion assay showing increased adhesion in LUCAT1-overexpressing cells compared to controls. (D) RT-qPCR analysis of epithelial-mesenchymal transition (EMT) markers CDH1 (E-cadherin) and CDH2 (N-cadherin) in sh-LUCAT1 and control cells. (E) Western blot analysis of E-cadherin and N-cadherin protein expression levels, confirming EMT suppression upon LUCAT1 knockdown. sh-NC: sh-negative control, sh-L: sh-LUCAT1. (*: p \u0026lt; 0.05, ***: p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/58572b5ca5ad740fa238481e.jpg"},{"id":89562369,"identity":"2d43f62e-15e6-4746-b40c-f0b0d67204f6","added_by":"auto","created_at":"2025-08-21 10:24:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3109004,"visible":true,"origin":"","legend":"\u003cp\u003eLUCAT1 silencing enhances therapeutic sensitivity in head and neck cancer (HNC) cells. (A) Clonogenic survival assay after 4 Gy irradiation shows reduced survival in sh-LUCAT1 cells, indicating enhanced radiosensitivity. (B) Cell viability assay following 48-hour cisplatin treatment (0–5 µg/mL), demonstrating increased chemosensitivity in LUCAT1-silenced cells. (C) Flow cytometric analysis of intracellular ROS levels using H₂DCF-DA staining after cisplatin exposure. ROS production was reduced in LUCAT1-overexpressing cells. (D) RT-qPCR and (E) western blot analyses of ROS-scavenging enzymes (GCLC, GCLM, GPX2) in sh-LUCAT1 and control cells, showing downregulation of these enzymes upon LUCAT1 knockdown. sh-NC: sh-negative control, sh-L: sh-LUCAT1, Gy: gray, CPT: camptothecin. (*: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001, n.s.: non-significant).\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/ab8ab3f9efb3b78af2fbb704.jpg"},{"id":89562167,"identity":"0ce9e2d9-cd55-4870-a9de-33451390bf4d","added_by":"auto","created_at":"2025-08-21 10:24:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5618054,"visible":true,"origin":"","legend":"\u003cp\u003eLUCAT1 mediates arecoline-induced malignant phenotypes in HNC cells. (A) Matrigel invasion assay showing increased invasiveness upon arecoline treatment in OECM1 and CGHNC8 cells; this effect is attenuated in LUCAT1-knockdown cells. (B) Wound-healing assay demonstrating that arecoline-induced migration is suppressed by LUCAT1 silencing. (C) Clonogenic survival assay showing that arecoline-induced radioresistance is attenuated in LUCAT1-knockdown cells following 4 Gy irradiation. (D) Cisplatin sensitivity assay shows reversal of arecoline-induced chemoresistance upon LUCAT1 silencing. Pt: parental cell line, Arc: arecoline subline, sh-NC: sh-negative control, sh-L: sh-LUCAT1, Gy: gray. (*: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001, n.s.: non-significant).\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/28793a465057f8368223886a.jpg"},{"id":89565282,"identity":"b7357df4-9942-41ef-bacd-2030a311fd01","added_by":"auto","created_at":"2025-08-21 10:40:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2362303,"visible":true,"origin":"","legend":"\u003cp\u003eLUCAT1 drives arecoline-induced EMT and antioxidant responses in HNC Cells. (A) RT-qPCR analysis and (B) western blot validation of EMT markers (CDH1, CDH2) reveal that arecoline induces EMT, while LUCAT1 knockdown reverses these changes. (C) RT-qPCR and (D) western blot analyses of antioxidant enzymes (GCLC, GCLM, GPX2) reveal that arecoline-induced expression of ROS-scavenging enzymes is suppressed by LUCAT1 knockdown. Pt: parental cell line, Arc: arecoline subline, sh-L: sh-LUCAT1, Gy: gray. (*: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001, n.s.: non-significant).\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/2ce1f169bfeb8ac2f03216d6.jpg"},{"id":89562334,"identity":"792f3cdd-df7f-4e8f-b297-3ad59e64dbef","added_by":"auto","created_at":"2025-08-21 10:24:31","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3895243,"visible":true,"origin":"","legend":"\u003cp\u003eLUCAT1 regulates cancer-associated pathways, including EMT and ROS clearance, via p38 MAPK activation in arecoline-treated HNC cells. (A) Scatter plot showing the differentially expressed genes (|fold change| ≥ 1.5) in LUCAT1-stable knockdown OECM1 and CGHNC9 cells compared to respective controls. A total of 290 genes were commonly upregulated (R2 region), and 503 genes were downregulated (R6 region) across both cell lines. (B) The top enriched KEGG pathways in OECM1 and CGHNC9 cells, regulated by LUCAT1 knockdown. (C) Gene Set Variation Analysis (GSVA) reveals enrichment of cancer-related pathways following LUCAT1 knockdown, including EGFR TKI resistance, VEGF signaling, ECM-receptor interaction, DNA damage response, p53 signaling, and glutathione metabolism. (D) Gene Set Enrichment Analysis (GSEA) comparing the LUCAT1-regulated gene signature (R2+R6) with the previously reported areca nut-induced transcriptomic profile [30], indicating significant correlation (NES = −1.197, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). The x-axis shows the ranked gene list; the y-axis indicates the enrichment score (ES). (E) Western blot analysis showing enhanced p38 MAPK phosphorylation in arecoline-treated OECM1 and CGHNC8 cells, which is attenuated upon LUCAT1 knockdown. (F) Functional validation using the p38 activator anisomycin. Treatment induces p38 phosphorylation, increases GPX2 expression, and decreases E-cadherin levels. These effects are abolished in LUCAT1-silenced cells, confirming LUCAT1’s role in mediating p38-dependent EMT and ROS clearance.\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/0c925308a055651e6f63a3c3.jpg"},{"id":89562298,"identity":"158da386-b2e7-4a52-97e2-3b5535dc1bf1","added_by":"auto","created_at":"2025-08-21 10:24:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":14295629,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic and bioinformatic analyses identify STAT1 and CUX1 as key transcription factors regulating LUCAT1 expression in arecoline-treated HNC cells. (A) DNase I hypersensitivity sites (DHSs) within the LUCAT1 promoter region (−2000 to +100 bp from the transcription start site) were identified using the ENCODE database. Three high-signal DHS clusters (chr5:91314302–91314570, chr5:91315480–91315630, and chr5:91315800–91315950) were defined as potential transcriptionally active regions. Transcription factor binding sites were predicted using the JASPAR database (PWM \u0026gt; 0.8, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), yielding 494 candidate LUCAT1-regulatory TFs. (B) Venn diagram showing integration of predicted TFs with a previously published arecoline-induced proteomic dataset, identifying three overlapping transcription factors: STAT1, IRF6, and CUX1. (C) Western blot analysis confirming transcription factor knockdown in HNC cells. (D) RT-qPCR quantification of LUCAT1 levels showing that silencing STAT1 or CUX1 significantly suppresses basal and arecoline-induced LUCAT1 expression, whereas IRF6 knockdown shows a weaker effect. Data are representative of at least three independent experiments. Pt: parental cell line, Arc: arecoline subline. (*: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001, n.s.: non-significant).\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/ba03ddc579c2b42167232299.jpg"},{"id":89562338,"identity":"a02613da-47b6-423e-9f5d-7c8a8f30e5d3","added_by":"auto","created_at":"2025-08-21 10:24:31","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3464305,"visible":true,"origin":"","legend":"\u003cp\u003eArecoline induces STAT1-mediated LUCAT1 expression through mAChR signaling in HNC cells. (A) Western blot analysis of total and phosphorylated STAT1 (p-STAT1) levels in HNC cells treated with increasing concentrations of the STAT1 inhibitor fludarabine (0–30 μM). (B) RT-qPCR analysis showing dose-dependent downregulation of LUCAT1 expression following fludarabine treatment. (C) Arecoline treatment induces STAT1 activation. Western blot showing increased total STAT1 and p-STAT1 levels in HNC cells exposed to arecoline. (D) ChIP-qPCR analysis of STAT1 binding to LUCAT1 promoter with control IgG or anti-STAT1 antibody. (E) Effect of the muscarinic acetylcholine receptor (mAChR) antagonist atropine (0–30 μM) on STAT1 expression. Atropine reduces both total and phosphorylated STAT1 levels and attenuates arecoline-induced STAT1 activation. (F) RT-qPCR showing that atropine treatment also reduces LUCAT1 expression in a dose-dependent manner, further supporting the mAChR-mediated regulation of the STAT1-LUCAT1 axis by arecoline. Pt: parental cell line, Arc, arecoline subline, Flu: fludarabine. (*: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/fbe9f763fd8dd5f9d3551e6e.jpg"},{"id":89562360,"identity":"84b3fc2c-6e17-411f-919a-4e431c919b17","added_by":"auto","created_at":"2025-08-21 10:24:33","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6665251,"visible":true,"origin":"","legend":"\u003cp\u003eLUCAT1 functions downstream of STAT1 to mediate arecoline-induced cell motility and therapeutic resistance in HNC cells. (A) Matrigel invasion assay showing that STAT1 silencing suppresses cell invasion in HNC cells, while LUCAT1 overexpression enhances invasion. Notably, LUCAT1 overexpression rescues the inhibitory effect of STAT1 knockdown. (B) Wound-healing assay showing similar effects on cell migration. STAT1 silencing impairs migration, which is restored by ectopic LUCAT1 expression. (C) Clonogenic survival assay following 4 Gy irradiation. STAT1 knockdown sensitizes cells to radiation, while LUCAT1 overexpression increases radioresistance. Overexpression of LUCAT1 reverses the enhanced radiosensitivity caused by STAT1 silencing. (*: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001, n.s.: non-significant).\u003c/p\u003e","description":"","filename":"Figure8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/099d1e159b6b5388dbd6570d.jpg"},{"id":89562324,"identity":"1ff5ddb0-2bbf-4ead-9ef4-ea6e2b3af208","added_by":"auto","created_at":"2025-08-21 10:24:30","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1562627,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic presentation of the mAChR–STAT1–LUCAT1 axis driving areca nut-induced malignant progression in HNC. Arecoline stimulates muscarinic acetylcholine receptor (mAChR) signaling, leading to STAT1 activation and transcriptional upregulation of LUCAT1. Elevated LUCAT1 expression facilitates p38 MAPK activation, promotes epithelial-mesenchymal transition (EMT), and enhances reactive oxygen species (ROS) clearance. These effects collectively drive increased cell motility and resistance to radiotherapy and chemotherapy. Pharmacological inhibition of STAT1 with fludarabine suppresses LUCAT1 expression, suggesting a potential therapeutic strategy for targeting areca nut-associated HNC progression.\u003c/p\u003e","description":"","filename":"Figure9.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/30c54aab4af2ceb567e6fa3d.jpg"},{"id":107927726,"identity":"e268660c-c4d2-4559-970d-6451b9d17661","added_by":"auto","created_at":"2026-04-27 16:02:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":45697281,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/5a0d2e19-208f-43fc-b0fa-933681c17388.pdf"},{"id":89562327,"identity":"76d20540-1164-4de5-827c-1a5185ea1e44","added_by":"auto","created_at":"2025-08-21 10:24:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22951,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/888773c1c8dce203ebd01e8a.docx"},{"id":89562310,"identity":"249dd3d7-53a9-4a7c-b1bb-e7b100f91cfc","added_by":"auto","created_at":"2025-08-21 10:24:30","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":47038,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/feee049fe5fad9435d66b0ca.xlsx"},{"id":89562225,"identity":"410912c5-5d59-460f-8888-6ee711a223b3","added_by":"auto","created_at":"2025-08-21 10:24:27","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20208,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/39a39e0e1cb9b5e13c544cf9.xlsx"},{"id":89562292,"identity":"c54737e2-287f-4354-a2b0-05fefa9d2677","added_by":"auto","created_at":"2025-08-21 10:24:28","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":98155,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/461c399dce92101444b5bf11.xlsx"},{"id":89562296,"identity":"8534ae9c-e695-4a7b-988b-d47c3bd69c58","added_by":"auto","created_at":"2025-08-21 10:24:29","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3678101,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryuncroppedGelsandBlotsimage.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6980917/v1/3b13d1364d40cc3e609b788c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"LUCAT1 Drives Arecoline-Induced Head and Neck Cancer Progression via STAT1-Mediated Transcriptional Regulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHead and neck cancer (HNC), encompassing malignancies of the lip, oral cavity, pharynx, and larynx, represents a significant global health challenge, with estimated 946,000 new cases and 482,000 deaths reported in 2022 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Standard treatments, including surgery, radiotherapy, and chemotherapy, are effective for early-stage HNC, achieving five-year survival rates exceeding 80% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, advanced-stage HNC is characterized by tumor invasion, local recurrence, and resistance to therapies such as cisplatin, resulting in significantly reduced survival rates [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These clinical challenges highlight the need to elucidate the molecular mechanisms driving HNC progression and therapeutic resistance.\u003c/p\u003e\u003cp\u003eHNC is associated with several etiological factors, including tobacco smoking, alcohol consumption, human papillomavirus (HPV) and Epstein\u0026ndash;Barr virus (EBV) infections, and areca nut chewing [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Areca nut chewing, a culturally entrenched practice in Southeast Asia, is a major etiological factor due to its potent carcinogenic properties [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Arecoline, the main alkaloid in areca nut, induces genotoxic and cytotoxic effects in HNC cells through mechanisms such as DNA damage, oxidative stress, and epithelial\u0026ndash;mesenchymal transition (EMT), all of which promote tumor progression [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Patients with a history of areca nut chewing often present with more aggressive tumor phenotypes, increased incidence of secondary primary tumors, reduced sensitivity to chemoradiotherapy, and poorer survival outcomes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite these observations, the molecular regulators mediating areca nut-induced HNC phenotypes remain poorly understood.\u003c/p\u003e\u003cp\u003eLong noncoding RNAs (lncRNAs), transcripts longer than 200 nucleotides with no protein-coding potential, have emerged as pivotal regulators in cancer biology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. By interacting with chromatin, RNA, or proteins, lncRNAs modulate transcriptional regulation, RNA stability, and signal transduction, influencing tumorigenesis processes such as proliferation, invasion, and cancer stemness [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although areca nut is a well-established carcinogen in HNC, the role of lncRNAs in areca nut-induced carcinogenesis remains underexplored, with limited studies identifying dysregulated lncRNAs requiring further validation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur previous transcriptomic analysis identified 28 lncRNAs associated with areca nut-induced HNC, with LUCAT1 (Lung Cancer Associated Transcript 1) showing significant upregulation in response to arecoline treatment [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. LUCAT1, also known as SCAL1, was initially reported to be elevated in the airway epithelia of cigarette smokers [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Subsequent studies have implicated LUCAT1 in multiple cancers, including lung, breast, liver, renal, and bladder cancers, where it promotes oncogenesis through mechanisms such as competitive binding to miRNAs (e.g., miR-4316/VEGF-A, miR-375/YAP1) or interactions with oncogenic proteins (e.g., IGF2BP2, HSP90) [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22 CR23\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, its role in areca nut-associated HNC remains uncharacterized.\u003c/p\u003e\u003cp\u003eThe present study investigates the mechanistic role of LUCAT1 in areca nut-induced HNC. We first delineate LUCAT1\u0026rsquo;s contribution to malignant phenotypes, including enhanced invasiveness and therapeutic resistance. We then demonstrate that arecoline upregulates Signal Transducer and Activator of Transcription 1 (STAT1), which transcriptionally activates LUCAT1. This arecoline\u0026ndash;STAT1\u0026ndash;LUCAT1 axis drives aggressive cancer phenotypes, including cell invasion, and cisplatin and radiation resistance. By elucidating this novel oncogenic pathway, our findings provide critical insights into areca nut-driven molecular carcinogenesis and identify LUCAT1 and STAT1 as potential targets for precision therapeutic strategies in areca nut-associated HNC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell Culture and Arecoline Treatment\u003c/h2\u003e\u003cp\u003eThree HNC cell lines, OECM1 (RRID: CVCL_6782), CGHNC8, and CGHNC9, were used in this study [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. OECM1 cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA), while CGHNC8 and CGHNC9 cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Thermo Fisher Scientific). Both media were supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific). Cells were incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO₂.\u003c/p\u003e\u003cp\u003eTo model the chronic effects of areca nut exposure, arecoline-adapted sublines were generated by treating parental cell lines with arecoline (Sigma-Aldrich, St. Louis, MO, USA) at an IC₃₀ concentration in complete medium for three months, as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These sublines were used for subsequent cellular and molecular analyses.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLUCAT1 and Related Gene Manipulation\u003c/h3\u003e\n\u003cp\u003eStable knockdown of LUCAT1 was achieved using CRISPR-based AAVS1 knock-in technology. A short hairpin RNA (shRNA) targeting exon 5 of LUCAT1 (shLUCAT1) was cloned into an AAVS1 knock-in donor vector (GeneCopoeia, Rockville, MD, USA), and an AAVS1-specific single-guide RNA (sgRNA) was subcloned into the pCas-Guide vector (OriGene Technologies, Rockville, MD, USA). These constructs, along with a scramble shRNA control, were co-transfected into HNC cell lines (OECM1 and CGHNC8) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). GFP-positive cells were sorted using a FACSAria III Cell Sorter (BD Biosciences, San Jose, CA, USA) and expanded by limiting dilution in the presence of puromycin (1 \u0026micro;g/mL) to establish single-cell clones. Stable LUCAT1-knockdown clones were subsequently treated with arecoline at an IC30 concentration for three months, as described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], to generate arecoline-adapted sublines.\u003c/p\u003e\u003cp\u003eFor LUCAT1 overexpression, the full-length LUCAT1 sequence (NCBI Reference Sequence: NR_103548.1) was cloned into the pcDNA3.1(\u0026minus;) vector (Thermo Fisher Scientific). The construct was transiently transfected into HNC cells using Lipofectamine 2000 for functional analyses. To investigate transcriptomic and transcription factor regulatory effects, small interfering RNAs (siRNAs) targeting LUCAT1, STAT1, CUX1, and IRF6 were transfected into HNC cells using Lipofectamine 2000, as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Transfections were performed according to the manufacturer\u0026rsquo;s protocol, with cells incubated at 37\u0026deg;C for 24 hours before downstream assays. Knockdown and overexpression efficiencies were validated by RT-qPCR. Oligonucleotide sequences are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eMigration, Invasion, and Adhesion Assays\u003c/h3\u003e\n\u003cp\u003eCell migration was evaluated using an in vitro wound healing assay, as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. HNC cells (4 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) were seeded in an ibidi culture-insert two-well system (ibidi, Gr\u0026auml;felfing, Germany). Once the cells attached, the inserts were removed and replaced with serum-free medium, and the gap area was photographed at regular intervals. Wound closure was quantified by measuring the gap area.\u003c/p\u003e\u003cp\u003eCell invasion was assessed using a Matrigel-coated transwell insert (Merck, Darmstadt, Germany) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. HNC cells (1.2\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were seeded into the upper chamber of inserts pre-coated with 5% Matrigel (Corning, Corning, NY, USA). The lower chamber contained medium with 20% FBS as a chemoattractant. After 18\u0026ndash;30 hours of incubation at 37\u0026deg;C, non-invading cells were removed from the upper surface. Invading cells on the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted under a microscope in five random fields per insert.\u003c/p\u003e\u003cp\u003eCell adhesion was measured using a solid-phase attachment assay. Ninety-six-well plates were pre-coated with 5% Matrigel and blocked with 0.5% bovine serum albumin (BSA) in serum-free medium. HNC cells (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e) were seeded into each well and incubated at 37\u0026deg;C for 30 minutes. Non-adherent cells were removed by washing with 0.1% BSA-containing serum-free medium. Attached cells were fixed with 4% formaldehyde and stained with 0.1% crystal violet; the stain was dissolved in 10% acetic acid for absorbance measurement at 595 nm.\u003c/p\u003e\n\u003ch3\u003eRadiosensitivity and Chemosensitivity assay\u003c/h3\u003e\n\u003cp\u003eRadiosensitivity was determined using a clonogenic survival assay, as previously described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. HNC cells (OECM1 and CGHNC8) were seeded into 35-mm dishes, irradiated with 4 gray (Gy) using a Gammacell 3000 (MDS Nordion, Ottawa, ON, Canada), and incubated at 37\u0026deg;C for 7\u0026ndash;10 days to allow colony formation. Colonies were fixed with 4% formaldehyde, stained with 0.1% crystal violet, and counted. Plating efficiency (PE) was calculated as the ratio of colonies formed to cells seeded in unirradiated controls. Survival fraction (SF) was determined by dividing the PE of irradiated cells by that of unirradiated controls.\u003c/p\u003e\u003cp\u003eChemosensitivity to cisplatin was evaluated using a Cell Counting Kit-8 (CCK-8) viability assay (Dojindo Laboratories, Mashiki-machi, Kumamoto, Japan), as previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. HNC cells (2,500 per well) were seeded into 96-well plates and allowed to adhere overnight at 37\u0026deg;C. Cells were treated with cisplatin (Sigma-Aldrich, St. Louis, MO, USA) at concentrations ranging from 0 to 5 \u0026micro;g/mL for 48 hours. CCK-8 reagent was added according to the manufacturer\u0026rsquo;s protocol, and absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated as the percentage of absorbance in treated cells relative to untreated controls.\u003c/p\u003e\n\u003ch3\u003eMeasurement of Intracellular ROS Levels\u003c/h3\u003e\n\u003cp\u003eIntracellular ROS levels were measured using the 5-(6)-Chloromethyl-2\u0026prime;,7\u0026prime;-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) assay (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s protocol. HNC cells were incubated with 1\u0026ndash;10 \u0026micro;M CM-H2DCFDA in PBS for 30 minutes at 37\u0026deg;C. Fluorescence intensity (excitation 488 nm, emission 525 nm) was measured using a Guava easyCyte Flow Cytometer (Cytek Biosciences, Fremont, CA, USA). Data analysis was performed using FlowJo software, with mean fluorescence intensity (MFI) used as the primary readout for ROS levels. Background fluorescence was determined using unstained cells. Results were expressed as normalization to the mode.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGene and Protein Expression Analysis\u003c/h2\u003e\u003cp\u003eGene expression was quantified using reverse transcription quantitative polymerase chain reaction (RT-qPCR), as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Total RNA was extracted from HNC cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Complementary DNA (cDNA) was synthesized using a reverse transcription kit (Bionovas, Toronto, ON, Canada) with 1 \u0026micro;g of RNA. RT-qPCR was performed using SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) on a CFX96 Real-Time PCR Detection System (Bio-Rad). Primer sequences for target genes, including LUCAT1, CDH1, CDH2, GCLC, GCLM, GPX2, STAT1, and GAPDH (reference gene), are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Relative gene expression was calculated using the 2⁻\u003csup\u003eΔΔCt\u003c/sup\u003e method, normalized to GAPDH.\u003c/p\u003e\u003cp\u003eProtein expression was analyzed by Western blotting, as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. HNC cells were lysed in CHAPS buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% CHAPS) and incubated on ice for 30 minutes. Lysates were centrifuged at 12,000 \u0026times; g for 15 minutes at 4\u0026deg;C to collect the supernatant. Protein was quantified via the Bradford assay (Bio-Rad, Hercules, CA, USA) followed by mixing with sample buffer and heating at 95℃ for 5 minutes. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Merck, Darmstadt, Germany). Membranes were blocked with 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween 20) and incubated overnight at 4\u0026deg;C with primary antibodies against target proteins, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Antibody details are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Merck) and imaged with an Amersham\u0026trade; Imager 600 (GE Healthcare). Band intensities were quantified using ImageJ software and normalized to GAPDH.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRNA sequencing\u003c/h3\u003e\n\u003cp\u003eTranscriptomic profiling was performed using RNA sequencing (RNA-seq) to compare differential gene expression between parental and LUCAT1 stable knockdown HNC cell lines (CGHNC9 and OECM1) and to elucidate LUCAT1\u0026rsquo;s downstream regulatory profile. Total RNA was extracted from HNC cells using TRIzol reagent according to the manufacturer\u0026rsquo;s protocol, and RNA integrity was assessed using the Caliper Labchip GX System (PerkinElmer, Waltham, MA, USA). Strand-specific RNA libraries were prepared with the TruSeq Stranded mRNA Sample Preparation kit (Illumina, San Diego, CA, USA), and library quality was verified using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Sequencing was conducted on a NextSeq 500 (Illumina, San Diego, CA, USA) with 150-bp paired-end reads. Raw RNA-seq data quality was confirmed using FastQC, and alignment was performed using the GDC mRNA analysis pipeline against GENCODE v43 (GRCh38.p13). Gene expression was quantified as TPM via the RSEM package (v1.3.3).\u003c/p\u003e\n\u003ch3\u003eChromatin immunoprecipitation followed by RT-qPCR (ChIP-qPCR)\u003c/h3\u003e\n\u003cp\u003eChromatin immunoprecipitation followed by RT-qPCR was based on the protocol provided by Abcam. Briefly, cells were fixed and cross-linked with formaldehyde and quenched with glycine. Nuclear fraction isolation was enriched by nuclear extraction buffer, and the chromatin was sheared by sonication to generate 150\u0026ndash;300 bp fragments. Following sonication, chromatin was incubated with anti-STAT1 or species-matched normal IgG antibody as a negative control, pre-coated on Qbeads-protein G (MagQu Co., Ltd., New Taipei City, Taiwan) overnight at 4℃. The targets were enriched and pulled down by antibody and detected by RT-qPCR. Data were analyzed using fold enrichment compared to IgG controls.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eBioinformatics Analysis\u003c/h2\u003e\u003cp\u003eBioinformatic analyses were performed on RNA sequencing (RNA-seq) data from parental and LUCAT1 stable knockdown HNC cell lines (CGHNC9 and OECM1) to investigate LUCAT1-mediated biological pathways and transcriptional regulation. Gene Set Variation Analysis (GSVA) was conducted using the GSVA R package (v1.50.5) to assess pathway activity across the transcriptomic dataset [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Gene Set Enrichment Analysis (GSEA) was performed using GSEA software (v4.2.3) to identify concordant differences in gene expression patterns between experimental groups.\u003c/p\u003e\u003cp\u003eTo investigate LUCAT1 transcriptional regulation, the LUCAT1 promoter region was defined as spanning \u0026minus;\u0026thinsp;2000 to +\u0026thinsp;100 base pairs relative to the transcription start site (TSS). DNase I hypersensitivity sites within this region were identified using ENCODE data accessed via the UCSC Genome Browser (GRCh38) to detect potential active regulatory elements. Transcription factor (TF) binding motifs were predicted using the JASPAR 2022 database, applying a position weight matrix (PWM) score threshold\u0026thinsp;\u0026gt;\u0026thinsp;0.8 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. TFs with predicted binding sites overlapping DNase I hypersensitivity regions were prioritized as candidate regulators of LUCAT1 transcription.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eData Processing and Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll experiments were conducted in at least triplicate unless otherwise specified. Data are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical comparisons between two groups were performed using two-tailed Student\u0026rsquo;s t-tests. For comparisons involving multiple groups or conditions, one-way or two-way analysis of variance (ANOVA) was used, followed by post-hoc Tukey\u0026rsquo;s tests for pairwise comparisons. Statistical analyses were performed using R (v4.3.3) and GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant, unless otherwise indicated (e.g., FDR for GSEA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eLUCAT1 Silencing Abrogates Cell Motility in HNC cells by inhibiting EMT\u003c/h2\u003e\u003cp\u003eTo elucidate the role of LUCAT1 in HNC progression, stable LUCAT1 knockdown cell lines (sh-LUCAT1) were generated in OECM1 and CGHNC8 cell lines using CRISPR-based AAVS1 knock-in technology. Cell motility phenotypes, including invasion, migration, and adhesion, were assessed using Matrigel invasion assays, wound-healing assays, and solid-phase attachment assays, respectively. LUCAT1 depletion significantly reduced cell invasion in OECM1 and CGHNC8 cells compared to scramble controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Similarly, migration was inhibited at 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In contrast, cell adhesion was enhanced following LUCAT1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), suggesting that LUCAT1 modulates cell-extracellular matrix (ECM) interactions. Given the critical role of EMT in cancer cell motility and invasion [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], EMT marker expression was examined. RT-qPCR analysis revealed increased \u003cem\u003eCDH1\u003c/em\u003e (E-cadherin) and decreased \u003cem\u003eCDH2\u003c/em\u003e (N-cadherin) mRNA levels in LUCAT1-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Western blotting confirmed these changes at the protein level, showing higher E-cadherin and lower N-cadherin expression in LUCAT1-silencing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These findings indicate that LUCAT1 promotes HNC cell motility by inducing EMT, and its silencing inhibits invasive phenotypes through EMT suppression.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eLUCAT1 Silencing Enhances Radio- and Chemosensitivity in HNC Cells via Modulation of ROS Homeostasis\u003c/h2\u003e\u003cp\u003eTo investigate the impact of LUCAT1 on therapeutic resistance, we assessed radiosensitivity and chemosensitivity in LUCAT1-knockdown OECM1 and CGHNC8. A clonogenic survival assay following 4 Gy irradiation revealed a reduction in surviving colonies compared to controls, indicating enhanced radiosensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Chemosensitivity to cisplatin was evaluated and presented increased cisplatin sensitivity, reducing cell viability in LUCAT1-knockdown cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Given that radiotherapy and chemotherapy induce DNA damage in part via reactive oxygen species (ROS) generation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], we investigated LUCAT1\u0026rsquo;s impact on ROS levels. HNC cells overexpressing LUCAT1 (via pcDNA3.1-LUCAT1) were treated with camptothecin (CPT), and ROS production was measured using the H\u003csub\u003e2\u003c/sub\u003eDCFDA assay. LUCAT1 overexpression significantly reduced ROS accumulation compared to vector controls following CPT treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To elucidate the mechanism, we analyzed ROS-scavenging enzymes (GCLC, GCLM, and GPX2) in LUCAT1-knockdown cells. RT-qPCR showed reduced mRNA levels of \u003cem\u003eGCLC\u003c/em\u003e, \u003cem\u003eGCLM\u003c/em\u003e, and \u003cem\u003eGPX2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), with Western blotting confirming decreased protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These results suggest that LUCAT1 contributes to therapeutic resistance in HNC cells by upregulating ROS-scavenging enzymes, and its silencing enhances sensitivity by disrupting ROS homeostasis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eLUCAT1 Knockdown Reverses Arecoline-Induced Invasive and Resistance Phenotypes in HNC Cells\u003c/h2\u003e\u003cp\u003eOur prior studies identified LUCAT1 as an arecoline-inducible lncRNA in HNC cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To determine whether LUCAT1 mediated arecoline-driven malignant phenotypes, we chronically treated stable LUCAT1-knockdown (sh-LUCAT1) and control OECM1 and CGHNC8 cells with arecoline (IC30 dose-adaption) and assessed cell motility and therapeutic sensitivity. Arecoline treatment increased cell invasion in control cells, but this effect was significantly attenuated in LUCAT1-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similarly, arecoline-enhanced migration was reduced to near baseline levels in LUCAT1-knockdown cells at 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). For therapeutic sensitivity, arecoline increased radioresistance in control cells, with higher surviving colonies post-4 Gy irradiation, but LUCAT1 knockdown abrogated this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Likewise, arecoline-induced cisplatin resistance was reversed, with LUCAT1-knockdown cells showing significantly lower viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eTo elucidate the molecular mechanisms underlying these phenotypes, we analyzed EMT markers and ROS-scavenging enzymes. Arecoline significantly decreased \u003cem\u003eCDH1\u003c/em\u003e (E-cadherin) mRNA and increased \u003cem\u003eCDH2\u003c/em\u003e (N-cadherin) mRNA in control cells, indicative of EMT induction, but these changes were reversed in LUCAT1-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Western blotting confirmed restored E-cadherin and reduced N-cadherin protein levels in LUCAT1-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). For therapeutic sensitivity, arecoline upregulated ROS-scavenging enzyme \u003cem\u003eGCLC\u003c/em\u003e, \u003cem\u003eGCLM\u003c/em\u003e, and \u003cem\u003eGPX2\u003c/em\u003e mRNA in control cells, but these increases were significantly suppressed in LUCAT1-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), with similar reductions in proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These findings demonstrate that LUCAT1 is critical for arecoline-induced malignant phenotypes in HNC cells. Specifically, LUCAT1 facilitates arecoline-driven cell motility by promoting EMT, as evidenced by altered E-cadherin and N-cadherin expression, and enhances therapeutic resistance by upregulating ROS-scavenging enzymes, thereby reducing intracellular chemotherapy-induced ROS levels. Silencing LUCAT1 effectively reverses these effects, underscoring its pivotal role in areca nut-associated HNC progression.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eLUCAT1 Facilitates EMT and Modulates ROS Clearance via p38 MAPK Signaling in Arecoline-Treated-HNC Cells\u003c/h2\u003e\u003cp\u003eTo elucidate LUCAT1\u0026rsquo;s downstream regulatory network in HNC, we performed RNA sequencing (RNA-seq) on control and LUCAT1-knockdown (si-LUCAT1) OECM1 and CGHNC9 cell lines. Genes with a |fold change| \u0026ge; 1.5 were identified, revealing 290 commonly upregulated and 503 commonly downregulated genes across both cell lines upon LUCAT1 silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Notably, CGHNC9 cells, derived from an areca nut chewer [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], exhibited more pronounced transcriptional changes than OECM1 cells, suggesting LUCAT1\u0026rsquo;s role in areca nut-associated gene dysregulation. Gene set variation analysis (GSVA) was conducted using the GSVA R package to assess pathway enrichment in LUCAT1-knockdown cells. Despite differences in baseline transcriptomes, both cell lines showed consistent enrichment patterns, including oncogenic signaling (e.g., EGFR tyrosine kinase inhibitor resistance, VEGF signaling), cell motility (e.g., ECM-receptor interaction), and stress response (e.g., DNA damage repair, p53 signaling, glutathione metabolism) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). These pathways aligned with our functional findings of enhanced motility, EMT, and therapeutic resistance (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). To evaluate LUCAT1\u0026rsquo;s role in areca nut-induced transcriptional reprogramming, we perform gene set enrichment analysis (GSEA) using GSEA software, comparing the shared LUCAT1-regulated gene set (up- and downregulated genes) with a published areca nut-induced downregulated gene set [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A significant negative correlation was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), indicating that LUCAT1 orchestrates areca nut-driven gene expression changes in HNC cells. The enrichment of glutathione metabolism, consistent with LUCAT1\u0026rsquo;s regulation of ROS-scavenging enzymes (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;E, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;D), prompted further investigation into oxidative stress mechanisms. Given that p38 MAPK signaling enhanced antioxidant enzyme expression to reduce intracellular ROS levels [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], we hypothesized that LUCAT1 modulated ROS via p38 MAPK activation. Arecoline treatment markedly increased phosphorylated p38 (p-p38) levels across both cell lines, while LUCAT1 knockdown significantly attenuated p-p38 levels with minimal effect on total p38 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). To validate p38\u0026rsquo;s role, we treated cells with anisomycin, a p38 activator [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Anisomycin increased p-p38, upregulated GPX2, and suppressed E-cadherin, mimicking arecoline\u0026rsquo;s effects, but these changes were significantly attenuated in LUCAT1-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These results demonstrate that LUCAT1 acts as an upstream regulator of p38 MAPK signaling in arecoline-treated HNC cells, promoting EMT and modulating ROS through enhanced expression of ROS-scavenging enzymes, thereby driving cancer progression via increased motility and oxidative stress tolerance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of STAT1 and CUX1 as Key Transcription Factors Regulating LUCAT1 Expression in Arecoline-Treated HNC Cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the transcriptional regulation of LUCAT1 in response to arecoline, we analyzed the LUCAT1 promoter region defined as the genomic interval spanning \u0026minus;\u0026thinsp;2000 to +\u0026thinsp;100 base pairs relative to the transcription start site, using the UCSC Genome Browser. Transcription factor (TF) binding sites were predicted using two complementary bioinformatics platforms: JASPAR 2022 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and ENCODE [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. JASPAR provided curated position weight matrices (PWMs) to identify high-confidence TF binding sites (PWM\u0026thinsp;\u0026gt;\u0026thinsp;0.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while ENCODE\u0026rsquo;s DNase I hypersensitivity site (DHS) data highlighted active regulatory regions. Three DHS-rich regions were identified within the LUCAT1 promoter: chr5:91314302\u0026ndash;91314570, chr5:91315480\u0026ndash;91315630, and chr5:91315800\u0026ndash;91315950 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). JASPAR analysis predicted 494 candidate TFs potentially binding these DHS-defined loci (Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). To prioritize TFs relevant to arecoline-induced LUCAT1 expression, we integrated the predicted TFs with a published areca nut-induced proteomic dataset identifying 1,083 differentially expressed proteins in areca nut-adapted HNC cell sublines [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Cross-referencing revealed three overlapped TFs\u0026mdash;STAT1, IRF6, and CUX1\u0026mdash;as potential regulators of LUCAT1 transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). To validate their regulation of LUCAT1, we performed siRNA-mediated knockdown of STAT1, IRF6, and CUX1 in OECM1 and CGHNC8 cells, with or without arecoline treatment (IC30 dose-adaption) and assessed LUCAT1 expression by RT-qPCR. Western blotting confirmed TF knockdown efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Silencing STAT1 or CUX1 significantly decreased LUCAT1 expression and attenuated arecoline-induced LUCAT1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In contrast, IRF6 knockdown modestly reduced LUCAT1 levels in arecoline-treated cells but increased LUCAT1 expression in control cells, suggesting a limited, context-dependent role (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings indicate that STAT1 and CUX1 are critical transcriptional activators of LUCAT1 in arecoline-treated HNC cells, with IRF6 exerting a minor regulatory influence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eSTAT1 Activation Induced LUCAT1 Expression in Response to Arecoline Exposure\u003c/h2\u003e\u003cp\u003ePrior studies have established that STAT1 serves as a transcription factor regulating oncogenic pathways in HNC [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], prompting further investigation of STAT1\u0026rsquo;s role in LUCAT1 expression. To confirm STAT1\u0026rsquo;s regulatory function, we treated OECM1 and CGHNC8 cells with fludarabine, a STAT1 inhibitor [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], at 0\u0026ndash;30 \u0026micro;M. Western blotting showed a dose-dependent reduction in total STAT1 and phosphorylated STAT1 (p-STAT1) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Correspondingly, RT-qPCR revealed a dose-dependent decrease in LUCAT1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), supporting STAT1\u0026rsquo;s role as a transcriptional activator of LUCAT1.\u003c/p\u003e\u003cp\u003eWe hypothesized that arecoline enhances STAT1 activation, thereby upregulating LUCAT1 expression. We validated that arecoline treatment (IC30 dose-adaption) increases total and p-STAT1 in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). To confirm if STAT1 regulates LUCAT1 expression via its promoter, we performed a ChIP assay. The result revealed that STAT1 indeed interacts with LUCAT1 promoter regions, especially DHS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Given that arecoline is a known muscarinic acetylcholine receptor (mAChR) agonist [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], we tested whether STAT1 activation is mAChR-dependent using atropine, an mAChR antagonist [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Atropine (200 \u0026micro;M) reduced total STAT1 and p-STAT1 levels, decreased both phosphorylated and total STAT1 levels, and significantly attenuated arecoline-induced STAT1 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). In parallel, LUCAT1 expression was significantly decreased with atropine treatment in both parental and arecoline-adapted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). These results demonstrate that arecoline induces LUCAT1 expression through mAChR-mediated STAT1 activation, establishing a mechanistic link between areca nut exposure and oncogenic LUCAT1 upregulation in HNC cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eThe STAT1-LUCAT1 Axis Drives Arecoline-Induced Invasive and Resistance Phenotypes in HNC\u003c/h2\u003e\u003cp\u003eTo confirm the functional interplay between STAT1 and LUCAT1, we investigated whether LUCAT1 overexpression could rescue the phenotypic effects of STAT1 silencing in OECM1 and CGHNC8 cells treated with arecoline (IC30 dose-adaptation). LUCAT1 overexpression increased invasion, while STAT1 silencing reduced invasion compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Importantly, ectopic LUCAT1 expression significantly restored the invasion ability in STAT1-silenced cells, indicating that LUCAT1 acts downstream of STAT1 in regulating invasiveness. Similarly, LUCAT1 overexpression enhanced migration, whereas STAT1 silencing decreased migration; LUCAT1 overexpression reversed this migration defect in STAT1-silenced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eFor radiosensitivity, LUCAT1 overexpression conferred resistance, increasing colony survival, while STAT1 silencing increased sensitivity by reducing survival colonies compared to control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Crucially, LUCAT1 overexpression counteracted the radiosensitizing effect of STAT1 silencing, restoring colony survival to near control levels. These findings demonstrate that LUCAT1 functions as a critical downstream effector of STAT1 in arecoline-treated HNC cells, mediating STAT1-driven invasive and resistant phenotypes. The STAT1-LUCAT1 regulatory axis is a central mechanism by which areca nut exposure promotes HNC progression, orchestrating enhanced cell motility and therapeutic resistance through arecoline-induced transcriptional activation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe escalating global burden of HNC, particularly in regions where areca nut chewing is prevalent, underscores the urgent need to elucidate the molecular mechanisms driving its progression [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In this study, we identified the lncRNA LUCAT1 as a critical downstream effector of areca nut-induced oncogenic signaling in HNC. We demonstrated that LUCAT1 promotes cell motility through the induction of EMT and enhances radio- and chemoresistance by modulating ROS homeostasis (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Knockdown of LUCAT1 reversed arecoline-induced invasive and resistant phenotypes, concomitant with attenuation of EMT markers and suppression of ROS-scavenging enzyme expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), partially mediated through the p38 MAPK signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Mechanistically, we discovered that LUCAT1 expression is transcriptionally regulated by STAT1, which is activated in response to arecoline stimulation (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, we established that arecoline-mediated STAT1 activation occurs via mAChR signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), thereby linking environmental carcinogen exposure to a defined transcriptional regulatory network. Functional rescue experiments confirmed that LUCAT1 acts downstream of STAT1 to drive arecoline-induced invasion, migration, and radioresistance in HNC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Collectively, these findings establish LUCAT1 as a pivotal lncRNA in arecoline-induced HNC progression, with the newly identified mAChR\u0026ndash;STAT1\u0026ndash;LUCAT1 signaling axis representing a promising molecular target for therapeutic intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLUCAT1 has been implicated in multiple cancers [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22 CR23\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Previous studies identified LUCAT1 as a driver of proliferation and metastasis in lung and breast cancers, in part through EMT induction [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Consistent with these reports, we demonstrate that LUCAT1 promotes cell motility and EMT in HNC, while its silencing suppresses invasion and migration, thus extending its oncogenic role to an arecoline-driven context. LUCAT1 has also been shown to promote chemoresistance and cancer stemness in lung, breast, and bladder cancers [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In line with these findings, we reveal that LUCAT1 enhances arecoline-induced radio- and chemoresistance in HNC and further demonstrate that LUCAT1 upregulates antioxidant enzymes such as GCLC, GCLM, and GPX2, thereby enhancing therapeutic resistance. These results establish a comprehensive mechanism by which areca nut promotes malignancy through LUCAT1 upregulation.\u003c/p\u003e\u003cp\u003eOur transcriptomic profiling further supported these observations, showing that the LUCAT1-associated mRNA signature closely aligns with the transcriptional changes induced by arecoline (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Functional pathway analysis revealed enrichment of stress-response pathways, including JAK\u0026ndash;STAT, cAMP signaling pathways, and glutathione metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). Previous studies reported that p38 MAPK activation under cellular stress conditions can induce antioxidant genes and facilitate EMT, invasion, and therapy resistance [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and that chronic areca nut exposure can activate the p38 pathway [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Our results are consistent with these findings, demonstrating that LUCAT1 promotes EMT and ROS modulation through p38 MAPK signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) and uniquely linking p38 regulation to LUCAT1 in arecoline-treated HNC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Thus, LUCAT1 functions upstream of p38 MAPK to promote cell motility and resistance to therapy via EMT induction and ROS clearance.\u003c/p\u003e\u003cp\u003eWhile arecoline has been previously associated with DNA damage, oxidative stress, and EMT induction in HNC cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], the specific transcriptional mediators of these carcinogenic effects were previously unknown. Here, we identify LUCAT1 as an arecoline-inducible lncRNA and further elucidate its upstream transcriptional regulators. Through integrated bioinformatic and experimental approaches, we found that STAT1, CUX1, and IRF6 potentially bind to the LUCAT1 promoter (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B) and are upregulated by arecoline exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Among these, STAT1 and CUX1 were validated as key transcriptional activators of LUCAT1, whereas IRF6, despite being induced by arecoline and implicated in inflammation-related lncRNA-mRNA networks [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], did not significantly regulate LUCAT1 expression in our system (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). The enrichment of the JAK\u0026ndash;STAT signaling pathway among LUCAT1-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) further pointed to STAT1 as a critical node warranting deeper investigation.\u003c/p\u003e\u003cp\u003eSTAT1 is classically known as a mediator of interferon signaling, capable of exerting both tumor-promoting and tumor-suppressive roles depending on the cellular context [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Recent studies, however, have demonstrated that STAT1 acts as an oncogenic driver in HNC, promoting cell proliferation, metastasis, and radioresistance [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Consistent with these observations, our data show that arecoline exposure upregulates STAT1 expression and phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), leading to the activation of LUCAT1-mediated malignant phenotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These results underscore the pivotal role of STAT1 as an upstream regulator of LUCAT1 under arecoline stimulation. Furthermore, we demonstrated that STAT1 activation by arecoline is dependent on muscarinic acetylcholine receptor (mAChR) signaling, as treatment with atropine\u0026mdash;an mAChR antagonist\u0026mdash;attenuated both total and phosphorylated STAT1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) and concomitantly reduced LUCAT1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Together, these findings establish a novel mechanistic link wherein arecoline activates the STAT1\u0026ndash;LUCAT1 axis via mAChR signaling, driving oncogenic transcriptional reprogramming and malignant progression in HNC.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study uncovers a novel mAChR\u0026ndash;STAT1\u0026ndash;LUCAT1 signaling axis that links arecoline exposure to transcriptional reprogramming, EMT induction, ROS scavenging, facilitates cell motility and therapy resistance in head and neck cancer. By demonstrating that LUCAT1 functions as a central downstream effector of STAT1-mediated oncogenic signaling, our findings provide a mechanistic framework that explains how an environmental carcinogen drives malignant transformation at the molecular level. These results not only expand the functional understanding of LUCAT1 and STAT1 in HNC but also establish this axis as a promising target for therapeutic intervention in areca nut-associated cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHNC: head and neck cancer; HPV: human papillomavirus; EBV: Epstein\u0026ndash;Barr virus; EMT: epithelial\u0026ndash;mesenchymal transition; ROS: reactive oxygen species; LncRNA: long noncoding RNA; LUCAT1: lung cancer-associated transcript 1; HSP90: heat shock protein 90; STAT: signal transducer and activator of transcription; YAP1: Yes1 Associated Transcriptional Regulator; MAPK: Mitogen-activated protein kinase; FBS: Fetal Bovine Serum; AAVS1: adeno-associated virus integration site 1; IC30: 30% maximal inhibitory concentration; CUX1: Cut Like Homeobox 1; IRF6: Interferon regulatory factor 6; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TPM: transcripts Per Million; RSEM: RNA-Seq by Expectation-Maximization; GSVA: gene Set Variation Analysis; GSEA: gene set enrichment analysis; PWM: Position weight matrix; ECM: extracellular matrix; CPT: camptothecin; GCLC: Glutamate\u0026ndash;cysteine ligase catalytic subunit ; GCLM: Glutamate\u0026ndash;Cysteine Ligase Modifier Subunit; GPX2: Glutathione Peroxidase 2; Pt: parental subline; Arc: arecoline subline; JAK: Janus kinase; TF: transcription factor; mAChR: muscarinic acetylcholine receptor; ATF2: Activating transcription factor 2\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that every dataset generated or analyzed during this study is either presented within this published article or can be obtained from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the Chang Gung Memorial Hospital–Linkou Medical Center (CMRPD1N0361).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHung-Han Huang:\u003c/strong\u003e Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Software, Validation, Visualization, Writing–original draft. \u003cstrong\u003eGuo-Rung You:\u003c/strong\u003e Conceptualization, Methodology, Software, Writing–review \u0026amp; editing. \u003cstrong\u003eJoseph T. Chang:\u003c/strong\u003e Funding acquisition, Project administration, Resources, Supervision, Writing–review \u0026amp; editing. \u003cstrong\u003eAnn-Joy Cheng:\u0026nbsp;\u003c/strong\u003eConceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing–review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-sequence and bioinformatics analyses were performed by Molecular Medicine Research Center and Bioinformatics Core Laboratory, Chang Gung University, Taiwan.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin.2024;74(3):229-63.\u003c/li\u003e\n\u003cli\u003eChow LQM. 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MYH9 facilitates cell invasion and radioresistance in head and neck cancer via modulation of cellular ROS levels by activating the MAPK-Nrf2-GCLC pathway. Cells.2022;11(18):2855.\u003c/li\u003e\n\u003cli\u003eXu M, Wang SY, Wang YC, Wu HX, Frank JA, Zhang Z, et al. Role of p38\u0026gamma; MAPK in regulation of EMT and cancer stem cells. BBA - Mol Basis Dis.2018;1864(11):3605-17.\u003c/li\u003e\n\u003cli\u003eGarg K, Kumar A, Kizhakkethil V, Kumar P and Singh S. Overlap in oncogenic and pro-inflammatory pathways associated with areca nut and nicotine exposure. Cancer Pathog Ther.2024;2(3):187-94.\u003c/li\u003e\n\u003cli\u003eThangjam GS and Kondaiah P. Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes. J Periodontal Res.2009;44(5):673-82.\u003c/li\u003e\n\u003cli\u003eSu YY, Chien CY, Luo SD, Huang TL, Lin WC, Fang FM, et al. Betel nut chewing history is an independent prognosticator for smoking patients with locally advanced stage IV head and neck squamous cell carcinoma receiving induction chemotherapy with docetaxel, cisplatin, and fluorouracil. World J Surg Oncol.2016;14.\u003c/li\u003e\n\u003cli\u003eMeissl K, Macho-Maschler S, Muller M and Strobl B. The good and the bad faces of STAT1 in solid tumours. Cytokine.2017;89:12-20.\u003c/li\u003e\n\u003cli\u003eShan FY, Shen S, Wang XX and Chen G. BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway. Int J Oncol.2023;62(4).\u003c/li\u003e\n\u003cli\u003eKnitz MW, Darragh LB, Bickett TE, Bhatia S, Bukkapatnam S, Gadwa J, et al. Loss of cancer cell STAT1 improves response to radiation therapy and promotes T cell activation in head and neck squamous cell carcinoma. Cancer Immunol Immunother.2022;71(5):1049-61.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"[email protected]","identity":"cellular-and-molecular-biology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmbl","sideBox":"Learn more about [Cellular \u0026 Molecular Biology Letters](http://cmbl.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/CMBL/default.aspx","title":"Cellular \u0026 Molecular Biology Letters","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lung Cancer Associated Transcript 1 (LUCAT1), Signal Transducer And Activator Of Transcription 1 (STAT1), areca nut, arecoline, head and neck cancer, Epithelial–mesenchymal transition (EMT), Reactive oxygen species (ROS), Therapeutic resistance, p38 MAPK signaling","lastPublishedDoi":"10.21203/rs.3.rs-6980917/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6980917/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAreca nut chewing is a major environmental risk factor for head and neck cancer (HNC), particularly in Southeast Asia. However, the molecular mechanisms that link areca nut exposure to malignant progression remain poorly understood. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of oncogenesis, but their role in areca nut-associated HNC remains unexplored.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe performed functional assays, transcriptomic profiling, and bioinformatic analyses to investigate the role of the lncRNA LUCAT1 in arecoline-treated HNC cells. Cell motility, epithelial\u0026ndash;mesenchymal transition (EMT), reactive oxygen species (ROS) levels, and therapeutic resistance were assessed following LUCAT1 knockdown or overexpression. We identified upstream regulators of LUCAT1 through promoter analysis, transcription factor knockdown, and pharmacological inhibition.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eLUCAT1 expression was significantly upregulated by arecoline exposure and promoted cell motility, EMT, ROS clearance, and resistance to radiotherapy and chemotherapy. Knockdown of LUCAT1 reversed these malignant phenotypes and suppressed antioxidant enzyme expression, partly through modulation of the p38 MAPK pathway. Transcriptomic and promoter analyses identified STAT1 as a key transcription factor activated by arecoline through muscarinic acetylcholine receptor (mAChR) signaling. Functional rescue experiments confirmed that LUCAT1 acts downstream of STAT1 to sustain arecoline-induced tumor aggressiveness.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur findings define a novel mAChR\u0026ndash;STAT1\u0026ndash;LUCAT1 regulatory axis that mediates areca nut-induced malignant progression in HNC. This study not only reveals a critical molecular pathway linking environmental carcinogen exposure to oncogenic transcriptional reprogramming but also highlights LUCAT1 as a promising target for therapeutic intervention in high-risk HNC patients.\u003c/p\u003e","manuscriptTitle":"LUCAT1 Drives Arecoline-Induced Head and Neck Cancer Progression via STAT1-Mediated Transcriptional Regulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 10:23:30","doi":"10.21203/rs.3.rs-6980917/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-07T13:16:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-02T13:19:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-26T12:50:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-23T13:06:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T10:12:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296186534861865328053163147233600716283","date":"2025-09-09T08:44:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299615605040649383956442023098372870925","date":"2025-09-08T14:49:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115410770944440916599247597812673810072","date":"2025-09-06T07:53:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147545943556034411887355739970763978084","date":"2025-09-04T14:35:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143276543428299418227296131778686684970","date":"2025-09-04T09:01:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50602214062339439322407386954434389163","date":"2025-09-04T08:29:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261736739717145537818682676010954916929","date":"2025-09-04T08:00:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-13T07:08:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-11T13:06:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular \u0026 Molecular Biology Letters","date":"2025-08-06T09:29:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"[email protected]","identity":"cellular-and-molecular-biology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmbl","sideBox":"Learn more about [Cellular \u0026 Molecular Biology Letters](http://cmbl.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/CMBL/default.aspx","title":"Cellular \u0026 Molecular Biology Letters","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8f24cc4d-647b-4f71-9f16-51472b3d4f1a","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:01:21+00:00","versionOfRecord":{"articleIdentity":"rs-6980917","link":"https://doi.org/10.1186/s11658-026-00925-9","journal":{"identity":"cellular-and-molecular-biology-letters","isVorOnly":false,"title":"Cellular \u0026 Molecular Biology Letters"},"publishedOn":"2026-04-22 15:57:59","publishedOnDateReadable":"April 22nd, 2026"},"versionCreatedAt":"2025-08-21 10:23:30","video":"","vorDoi":"10.1186/s11658-026-00925-9","vorDoiUrl":"https://doi.org/10.1186/s11658-026-00925-9","workflowStages":[]},"version":"v1","identity":"rs-6980917","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6980917","identity":"rs-6980917","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}