The RNA-binding protein La/SSB drives head and neck squamous cell carcinoma progression through TFAP2C-mediated transcriptional activation of FSCN1

The RNA-binding protein La/SSB drives head and neck squamous cell carcinoma progression through TFAP2C-mediated transcriptional activation of FSCN1 | 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 Article The RNA-binding protein La/SSB drives head and neck squamous cell carcinoma progression through TFAP2C-mediated transcriptional activation of FSCN1 Shiyin Ma, Shixian Liu, Deshang Chen, Wentao Zhang, Hui Li, Jie Peng, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8395850/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is marked by aggressive behavior, a paucity of effective treatment strategies, and unsatisfactory survival rates. The RNA-binding protein La/SSB is frequently overexpressed in diverse cancers, but its biological function and mechanistic contribution to HNSCC pathogenesis remain unclear. Methods: By combining transcriptome-wide RNA sequencing and chromatin accessibility analyses with functional experiments conducted both in vitro and in vivo, we sought to define the role of La/SSB in HNSCC. Patient-derived organoids (PDOs), cell-derived xenografts (CDXs), and conditional La/SSB knockout (La/ssb cKO ) mice were used to validate its tumorigenic and therapeutic relevance. Results: La/SSB expression was substantially elevated in HNSCC tissues and cell lines, with higher levels being associated with reduced overall survival. Functionally, La/SSB promoted tumor cell proliferation, invasion, and resistance to cisplatin. Integrative multi-omics analysis identified FSCN1 as a key downstream effector transcriptionally activated by TFAP2C. Mechanistically, La/SSB enhanced TFAP2C recruitment and chromatin accessibility at the FSCN1 promoter, thereby establishing a La/SSB–TFAP2C–FSCN1 regulatory axis that sustains oncogenic transcriptional programs. Inhibition or genetic deletion of La/SSB significantly sensitized tumors to cisplatin both in PDOs and La/ssb cKO mice. Conclusions: The La/SSB–TFAP2C–FSCN1 axis promotes HNSCC progression and chemoresistance by coupling RNA-binding activity with transcriptional reprogramming. Therapeutic modulation of La/SSB may offer a promising approach to improve cisplatin responsiveness in the management of HNSCC. Biological sciences/Cancer/Head and neck cancer Biological sciences/Cell biology/Mechanisms of disease HNSCC La/SSB TFAP2C FSCN1 tumor progression cisplatin resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Head and neck squamous cell carcinoma (HNSCC) represents the most prevalent histological subtype of head and neck cancers, accounting for more than 90% of cases[ 1 ]. Notwithstanding ongoing improvements in surgical approaches, radiotherapy, and the addition of immunotherapy, the five-year overall survival rate remains below 65%[ 2 – 4 ]. Local recurrence, distant metastasis, and resistance to chemotherapy—particularly to platinum-based regimens—remain major obstacles to successful treatment[ 5 , 6 ]. The molecular mechanisms underlying HNSCC progression and therapy resistance therefore warrant in-depth investigation to identify new prognostic markers and therapeutic targets. The La antigen, also known as Sjögren syndrome type B antigen (La/SSB or LARP3), is an evolutionarily conserved RNA-binding protein that participates in RNA maturation, stability, and translation[ 7 – 9 ]. Beyond its physiological roles in RNA metabolism, aberrant overexpression of La/SSB has been reported in multiple malignancies, including hepatocellular carcinoma, lung cancer, and breast cancer[ 10 – 12 ]. Elevated La/SSB levels are often associated with enhanced tumor proliferation, invasion, and resistance to genotoxic stress, suggesting that La/SSB may function as an oncogenic factor in cancer biology[ 13 – 15 ]. However, the specific role and regulatory mechanisms of La/SSB in HNSCC remain poorly understood. Recent studies have underscored the importance of RNA-binding proteins as molecular hubs that connect post-transcriptional regulation with chromatin remodeling and signal transduction, thereby reprogramming cellular phenotypes during malignant transformation[ 16 , 17 ]. Whether La/SSB engages in similar regulatory cross-talk to modulate tumor progression in HNSCC has not been systematically examined. In this study, we comprehensively investigated the expression pattern, functional significance, and mechanistic basis of La/SSB in HNSCC using integrated transcriptomic and chromatin accessibility profiling. Through in vitro and in vivo validation—including patient-derived organoids (PDOs), cell-derived xenografts (CDXs), and conditional La/SSB knockout mouse models—we demonstrate that La/SSB exerts a central influence on tumor proliferation, invasive capacity, and resistance to chemotherapy. Collectively, these data position La/SSB as a candidate prognostic indicator and a tractable therapeutic vulnerability in HNSCC. METHOD Animal Healthy BALB/c nude mice were sourced from GemPharmatech. (Jiangsu, China) and housed at the Animal Experiment Center of Anhui Medical University under conventional laboratory settings. All animal experiments were performed following protocols approved by the Ethics Committee of Anhui Medical University. (Anhui, China). For CDX experiments, six-week-old male BALB/c nude mice were randomly assigned to experimental groups (n = 5 per group) and subjected to different treatments. To evaluate the in vivo roles of La/SSB and FSCN1, 4 × 10⁶ cells from the indicated cell lines were resuspended in a volume of 0.2 mL PBS and subcutaneously administered into the axilla to generate xenograft tumors. Tumor size was monitored at 3-day intervals, tumor volume was determined using the formula: volume = (length × width²)/2 (mm³). At the end of the experiment, mice were euthanized, and tumors were harvested, weighed, and photographed, followed by immunohistochemical (IHC) assessment. For metastasis studies, nude mice received tail vein injections of 1 million genetically engineered cells suspended in 100 μL PBS. Eight weeks later, the mice were euthanized, and lung tissues were harvested for further analysis. Metastatic nodules were visualized under a dissecting microscope following hematoxylin and eosin (H&E) staining. For conditional knockout mice model, La/ssb fl/fl mice were crossed with Krt14 Cre/ERT2 mice to generate La/ssb fl/fl ; Krt14 Cre/ERT2 offspring. All mouse strains were sourced from the Shanghai Model Organisms Center and maintained under specific pathogen-free (SPF) conditions. To establish an HNSCC model, six-week-old La/ssb wt/wt ; Krt14 Cre/ERT2 mice (designated as La/ssb Ctrl ) and La/ssb fl/fl ; Krt14 Cre/ERT2 mice (designated as La/ssb cKO ) were administered drinking water supplemented with 4-nitroquinoline 1-oxide (4NQO, 50 μg/mL) for 16 weeks, followed by replacement with normal drinking water to allow tumor progression. For lineage tracing and conditional deletion of La/SSB, tamoxifen was delivered via intraperitoneal injection at a dose of 120 mg/kg every other day for a total of four injections to induce Cre recombinase activity. Additionally, mice received intraperitoneal injections of cisplatin (CDDP, 5 mg/kg) once per week or saline twice per week for four consecutive weeks. Upon completion of all treatments, mice were sacrificed, and tongue tissues were harvested for downstream analyses. Cell lines and culture conditions The human head and neck squamous cell carcinoma (HNSC) cell line FaDu and HEK-293T cells were obtained from the American Type Culture Collection (ATCC; VA, USA). The TU686 HNSCC cell line was obtained from the BeNa Culture Collection (Beijing, China; catalogue number BNCC359450), while TU212 (catalogue number HTX2130) and human normal oral keratinocytes (NOK; catalogue number HTX2992) were supplied by Otwo Biotech (Shenzhen, China). The HNSCC cell lines TU177 and LIU-LSC-1 were characterized in previous studie[18]. All cells were maintained in RPMI 1640 medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA) and penicillin–streptomycin (100 U/mL and 100 μg/mL, respectively; Beyotime, Jiangsu, China). Cells were cultured at 37°C in a humidified incubator with 5% CO₂ and routinely screened for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, #LT07-118). Additional details regarding cell sources and culture conditions are provided in Supplementary Table S4 . Sample collection Human HNSC specimens were obtained from patients treated at the First Affiliated Hospital of Anhui Medical University (Hefei, Anhui) between 2020 and 2025. Histopathological diagnoses were independently verified by a minimum of two experienced pathologists, and informed written consent was obtained from all participants before sample collection. All surgical and experimental procedures involving human tissues were performed in accordance with protocols approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University. Detailed clinicopathological characteristics, including patient age, sex, histological grade, TNM classification, lymph node status, and local invasion, are summarized in Supplementary Tables S3 and S5 . Clinical information for patients used in the establishment of PDO models is presented in Supplementary Table S6 . Antibodies and reagents Detailed information regarding all antibodies utilized in this study is listed in Supplementary Table S7. CDDP was sourced from MedChemExpress (Monmouth Junction, NJ, USA). Puromycin and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (MO, USA), whereas 4NQO was obtained from Santa Cruz Biotechnology (CA, USA). Quantitative real-time PCR (qRT-PCR) Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA). RNA quantity and purity were assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). For cDNA synthesis, 1 μg of total RNA was reverse-transcribed into first-strand cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative real-time PCR (qRT-PCR) was performed on a LightCycler 96 system (Roche, Switzerland) using SYBR Premix Ex Taq II (TaKaRa, Kyoto, Japan) following the manufacturer’s protocols. Relative expression levels of target mRNAs were determined using the 2^−ΔΔCt method, with β-actin serving as the reference gene. All primers were designed and synthesized by Sangon Biotech (Shanghai, China) and are provided in Supplementary Table S8 . H&E and immunohistochemical staining (IHC) Tumor tissues from various treatment groups or organoid models were fixed in formalin, embedded in paraffin, and sectioned for histological examination. After deparaffinization and rehydration, antigen retrieval was performed, followed by incubation with 3% hydrogen peroxide for 1 h to block endogenous peroxidase activity. Sections were then incubated with 3% bovine serum albumin for 1 h and subsequently with primary antibodies at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies were applied, and immunoreactivity was visualized using DAB substrate (Beyotime), with hematoxylin serving as a nuclear counterstain. Immunohistochemical staining was semi-quantitatively evaluated using H-scores, calculated as follows: [(percentage of weak staining × 1) + (percentage of moderate staining × 2) + (percentage of strong staining × 3)], resulting in scores ranging from 0 to 300. Western blotting Total cellular proteins were extracted using RIPA lysis buffer (Beyotime). Protein samples were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibodies (1:1000). Following washes, membranes were treated with the appropriate secondary antibodies at room temperature for 1 h. Protein signals were visualized using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, MA, USA) and captured with a ChemiScope 6100 imaging system (Clinx, Shanghai, China). Immunoffuorescence (IF) assay For immunofluorescence studies, cells were fixed with 4% formaldehyde, permeabilized using 0.5% Triton X-100 (Sigma-Aldrich), and blocked with Immunol Staining Blocking Buffer (Beyotime). Cells were incubated overnight with primary antibodies and subsequently treated with fluorophore-conjugated secondary antibodies (Cell Signaling Technology, MA, USA) for 1 h. Finally, cells were mounted using ProLong Gold Antifade Mountant containing DAPI (Thermo Fisher Scientific) before imaging. For multiplex immunofluorescence analysis of PDOs and mouse HNSC tissues, sections were stained with antibodies against La/SSB, Ki-67, and cleaved caspase-3. Immunoreactivity was detected using FITC- and Cy3-conjugated secondary antibodies (Cell Signaling Technology, MA, USA), followed by nuclear counterstaining with DAPI (Beyotime). To assess Ki-67 and cleaved caspase-3 expression, a minimum of three sections per HNSCC lesion were evaluated. In each section, more than 150 tumor cells were manually counted, and the numbers of Ki-67– or cleaved caspase-3–positive cells were recorded. The proportion of marker-positive cells was determined by dividing the number of positive cells by the total number of tumor cells, and the mean value across all sections was calculated. Lentivirus infection All lentiviral constructs were purchased from GenePharma (Shanghai, China), including LV4 plasmids encoding La/SSB, FSCN1, and TFAP2C cDNAs, as well as an empty vector control. In addition, the LV-2 N lentiviral shRNA vector was employed to knock down La/SSB, FSCN1, and TFAP2C, alongside a scrambled shRNA control (shSc). Target sequences are provided in Supplementary Table S9 . Lentiviral production and the establishment of stable cell lines were performed as described previously[19]. Cell viability HNSCC cells were seeded into 96-well plates at 1.5 × 10³ cells per well. Cell viability was assessed at 24, 48, 72, and 96 hours using the Cell Counting Kit-8 (CCK-8; Topscience, Shanghai, China). After a 2-hour incubation with the CCK-8 reagent at 37°C, absorbance was recorded at 450 nm to determine the optical density (OD). Colony formation assay Adherent cells were harvested and counted prior to seeding. Approximately 1,000 cells were plated per well in 6-cm culture dishes in triplicate and allowed to grow for 14–16 days. After incubation, the colonies were washed with PBS, fixed in 4% paraformaldehyde, and stained with 0.5% crystal violet (Beyotime, Jiangsu, China). Excess dye was gently washed away with water. Only colonies comprising more than 50 cells were counted and included in subsequent analyses. EdU staining assays For EdU incorporation assays, 4 × 10⁴ cells were plated in each well of 24-well plates and treated with 50 μM EdU for 2 h at 37°C. Following fixation and permeabilization, EdU labeling was performed using the Cell-Light EdU Apollo488 Kit (RiboBio, Guangzhou, China) according to the manufacturer’s protocol. Images were acquired using an LSM880+225 Airyscan confocal microscope (Carl Zeiss). The proliferation rate was calculated as the proportion of EdU-positive cells relative to DAPI-stained nuclei. Cell migration and invasion assays Transwell assays were conducted to evaluate cell migration and invasion. For invasion experiments, the upper surface of the Transwell inserts was coated with 40 μL of Matrigel (Corning, NY, USA). A total of 2 × 10⁴ cells in 200 μL of serum-free medium were added to the upper chamber, while the lower chamber was filled with medium containing 20% FBS. After 24 hours of incubation at 37°C, cells on the lower side of the membrane were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified under a microscope. Cell apoptosis analysis Cell apoptosis was evaluated using the Annexin V-APC/PI Apoptosis Detection Kit (Keygen, Jiangsu, China) according to the manufacturer’s instructions. In brief, 3 × 10⁵ adherent cells were harvested with EDTA-free trypsin and washed twice with PBS. Cells were resuspended in 500 μL Binding Buffer and incubated with 5 μL Annexin V-APC and 5 μL propidium iodide (PI) in the dark for 10 minutes. The percentage of apoptotic cells was determined by flow cytometry (Beckman Coulter, CA, USA). Establishment and culture of HNSCC organoids HNSCC organoids were generated and cultured with minor modifications to previously reported protocols. Fresh HNSCC tumor specimens were washed three times with ice-cold PBS (5 minutes per wash) and cut into 1–3 mm³ pieces on ice. The tissue fragments were digested with trypsin (Sigma-Aldrich) for about 30 minutes. Once the suspension became turbid, it was passed through a 100-μm cell strainer and centrifuged at 200 × g for 5 minutes to collect cell clusters. The collected cell clusters were washed three times with PBS to remove residual enzymes and then embedded in Matrigel (Corning). After the Matrigel had solidified, HNSCC organoid culture medium (BioGenous, Jiangsu, China) was added, and cultures were maintained at 37°C in a CO₂ incubator, with medium refreshed every 3–5 days. For lentiviral transduction, HNSCC organoids were first dissociated into small cell clusters using a pipette. The clusters were collected by centrifugation at 200 × g for 5 minutes at 4°C and resuspended in culture medium (BioGenous) containing the designated lentiviral particles. The suspension was subjected to spin infection at 700 × g for 90 minutes at 25°C, followed by incubation at 37°C for 4 hours. After a brief centrifugation at 300 × g for 5 minutes, the cell clusters were re-embedded in Matrigel. Organoid morphology was imaged, and diameters were measured using image analysis software (Photoshop, CA, USA). RNA sequencing RNA sequencing (RNA-seq) was performed by LC Sciences (Hangzhou, China). Briefly, cDNA was converted to double-stranded DNA, followed by PCR amplification and 2 × 150 bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000. The resulting reads were aligned and assembled using StringTie with default parameters to generate FPKM values. Differential gene expression between shLa/SSB FaDu cells and their control counterparts was analyzed using the edgeR package in R. Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) ATAC-seq was carried out with support from Igenebook (Wuhan, China). Nuclei were isolated from 50,000 cells, washed, and resuspended in nuclear lysis buffer. Chromatin accessibility was assessed via transposition using Tn5 transposase, which fragments and tags open chromatin regions. The transposed DNA was purified using the Qiagen MinElute PCR Purification Kit (Qiagen, Hilden, Germany) and amplified by PCR with unique barcodes. PCR products were further cleaned to remove residual primers and adapter dimers, and library quality and concentration were checked using an Agilent Bioanalyzer. Sequencing was performed on an Illumina NovaSeq™ platform to obtain 150 bp paired-end reads. Raw sequencing data were processed using a standard bioinformatics pipeline, including alignment, peak calling, and differential chromatin accessibility analysis. Reporter constructs and luciferase reporter assay A 190-bp fragment of the human FSCN1 gene (-1339/-1139), which includes the TFAP2C-binding site, was amplified from human genomic DNA by PCR. The resulting PCR product was inserted into the pGL4.23[luc2/minP] luciferase reporter vector (Promega) using homologous recombination. The primers used for amplification are listed below: forward, 5'-TAACTGGCCGGTACCGTTCTGGGGCTCAAGGCCCT-3'; reverse, 5'-CTTGATATCCTCGAGGGCCGGGCACTGAGATAACT-3'. A mutant reporter plasmid (pFSCN1^mut-Luc) was generated by altering the TFAP2C-binding site using the Q5 Site-Directed Mutagenesis Kit (NEB, MA, USA) with the following primers: forward, 5'-GGGCTAACACAGGCTCGGACCAGC-3'; reverse, 5'-CATTTAATCCCAGCCAGGGGCTTTC-3'. HEK-293T cells were seeded in 24-well plates and co-transfected with 200 ng of either FSCN1 wt -Luc or pFSCN1^mut-Luc, 200 ng of pcDNA3.1-TFAP2C or empty pcDNA3.1 vector, and 10 ng of the internal control plasmid pRL-TK. Luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega), and firefly luciferase signals were normalized to Renilla luciferase. Chromatin immunoprecipitation (ChIP) The binding of TFAP2C to the La/SSB gene was assessed by ChIP using a TFAP2C-specific antibody. ChIP assays were carried out following the protocol provided with the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, MA, USA). Primers used for PCR and qPCR to amplify the predicted TFAP2C-binding sites within the human La/SSB locus are listed in Supplementary Table S10 . Bioinformatics analysis RNA-seq data and corresponding clinical information for HNSC patients were obtained from The Cancer Genome Atlas (TCGA, http://cancergenome.nih.gov/). Survival analyses were conducted using Kaplan–Meier curves and the log-rank test. Patients in the TCGA-HNSC cohort were stratified into high- and low-expression groups according to the median expression levels of TFAP2C, La/SSB, or FSCN1. Gene expression correlations were assessed by Spearman’s correlation, and receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value of these genes. Statistical analysis Data are presented as mean ± standard deviation (SD). For comparisons between two groups, Student’s t-test was used, whereas one-way or two-way ANOVA was employed for analyses involving multiple groups. Statistical analyses were performed using GraphPad Prism 9.4.1. P values less than 0.05 were considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Results 1. La/SSB is upregulated in HNSCC and predicts poor patient prognosis Pan-cancer analysis of TCGA data indicated that La/SSB expression was markedly increased in various tumor types, including HNSCC (Fig. S1A). To clarify its clinical significance, La/SSB expression was compared between tumor and adjacent nonmalignant (ANM) tissues using TCGA, GSE127165, and GSE178537 datasets. All datasets consistently demonstrated marked upregulation of La/SSB in HNSCC tissues (Fig. 1A–C). Kaplan–Meier survival analysis demonstrated that patients with higher La/SSB expression had shorter overall survival (OS) and disease-specific survival (DSS) (Fig. 1D–E; Tables 1–2). Western blot analysis of 12 paired HNSCC and ANM samples revealed that La/SSB protein levels were elevated in the majority of tumor tissues (Fig. 1F). IHC staining of an expanded cohort of 60 HNSCC specimens further verified this finding, with significantly higher H-scores observed in tumor tissues compared with adjacent mucosa. Importantly, La/SSB levels were higher in tumors with advanced T stage (T3/T4 compared with T1/T2; Fig. 1G–H) and were associated with reduced overall survival (OS) (Fig. 1I). At the cellular level, quantitative RT-PCR and western blotting revealed substantially higher La/SSB expression in HNSCC cell lines (FaDu, TU177, TU686, TU212, LIU-LSC-1) compared with NOK (Fig. 1J). Immunofluorescence imaging confirmed predominant nuclear localization of La/SSB in HNSCC cells (Fig. 1K). Taken together, these findings support the role of La/SSB as an oncogenic biomarker that is linked to tumor progression and poor clinical outcomes in HNSCC. 2. La/SSB enhances proliferation, migration, and apoptosis resistance of HNSCC cells in vitro To investigate the functional consequences of La/SSB dysregulation, loss- and gain-of-function experiments were performed. Stable knockdown of La/SSB was achieved in FaDu cells using shRNAs (shLa/SSB#1 and shLa/SSB#2), whereas TU177 cells were engineered to overexpress La/SSB. Western blotting and qRT-PCR analyses confirmed the effective modulation of La/SSB expression (Fig. 2A–B, Fig. S2A–B). CCK-8, colony formation, and EdU assays demonstrated that depletion of La/SSB substantially inhibited the proliferation of HNSCC cells, while its overexpression significantly enhanced growth (Fig. 2C–D, Fig. S2C–E). Given the ability of PDOs to recapitulate tumor heterogeneity, we next evaluated La/SSB function in HNSCC PDO models. Consistent with the cell line data, organoids derived from La/SSB-overexpressing cells displayed larger diameters and stronger Ki-67 staining, whereas La/SSB depletion inhibited growth and reduced proliferative indices (Fig. 2E–I). Transwell assays showed that silencing La/SSB markedly impaired the migratory and invasive abilities of FaDu cells, whereas overexpression enhanced these phenotypes in TU177 cells (Fig. 2J–K). Flow cytometric analysis further showed that La/SSB silencing increased apoptotic cell populations, while its overexpression conferred apoptosis resistance (Fig. S2F–G). Together, these findings establish La/SSB as a potent promoter of HNSCC cell proliferation, migration, and survival in vitro. 3. La/SSB promotes tumor growth and metastasis in vivo To investigate the tumor-promoting function of La/SSB in vivo, CDX models were generated in nude mice using FaDu cells with stable La/SSB knockdown or TU177 cells overexpressing La/SSB. Tumor growth was monitored over four weeks. Silencing La/SSB significantly delayed tumor growth and reduced tumor volume and weight, whereas La/SSB overexpression markedly accelerated tumor progression (Fig. 3A–D; Fig. S3A–D). IHC analysis revealed lower Ki-67 and higher cleaved caspase-3 levels in La/SSB-deficient tumors, whereas the opposite pattern was observed in La/SSB-overexpressing xenografts (Fig. 3E–H; Fig. S3E–H). Furthermore, tail vein injection assays demonstrated that La/SSB knockdown dramatically decreased the number of pulmonary metastatic nodules, whereas La/SSB overexpression enhanced lung colonization (Fig. 3I–L). These results confirm that La/SSB promotes tumor growth and metastasis of HNSCC in vivo. 4. Integrated transcriptomic and epigenomic profiling identifies FSCN1 as a downstream effector of La/SSB To explore the molecular mechanisms driving La/SSB-mediated tumorigenesis, RNA sequencing (RNA-seq) and ATAC sequencing (ATAC-seq) were performed in FaDu cells after La/SSB knockdown. RNA-seq analysis revealed 2,813 differentially expressed genes (DEGs) with |log₂FC| > 1 and p < 0.05, including 1,521 downregulated and 1,292 upregulated transcripts (Fig. 4A; Table S1). ATAC-seq revealed substantial changes in chromatin accessibility after La/SSB depletion, with 7,973 peaks differentially enriched (Fig. 4B; Table S2). Integrative analysis of downregulated genes and regions with reduced chromatin accessibility, combined with upregulated genes from the TCGA-HNSC dataset, yielded seven overlapping candidates (Fig. 4C). Among these, FSCN1 expression was most strongly correlated with poor patient survival (p < 0.01; Fig. 4G, Fig. S4A–F). Consistent with these findings, FSCN1 mRNA expression was markedly elevated in HNSCC tissues compared with ANM across both TCGA and GEO datasets (Fig. 4D–F). ATAC-seq tracks visualized using Integrative Genomics Viewer (IGV) indicated markedly reduced chromatin accessibility at the FSCN1 promoter following La/SSB knockdown (Fig. 4H). Correspondingly, In FaDu cells with La/SSB silencing, FSCN1 mRNA and protein levels were decreased, whereas in TU177 cells overexpressing La/SSB, FSCN1 expression was elevated (Fig. 4I–J). IHC staining of clinical samples confirmed co-upregulation of La/SSB and FSCN1, Higher FSCN1 expression was correlated with advanced T stage and reduced overall survival (Fig. 4K–L). Collectively, these results identify FSCN1 as a key downstream effector transcriptionally regulated by La/SSB in HNSCC. 5. FSCN1 mimics and mediates the oncogenic functions of La/SSB in HNSCC To evaluate whether FSCN1 independently exhibits oncogenic activity similar to that of La/SSB, we first generated HNSCC cell lines with stable FSCN1 knockdown or overexpression (Fig. S5A). In FaDu cells, knockdown of FSCN1 significantly suppressed cell proliferation, colony formation, and DNA synthesis, as shown by CCK-8, colony formation, and EdU assays, whereas overexpression of FSCN1 in TU177 cells led to enhanced proliferation and DNA synthesis. (Fig. S5B–E). In PDO models, FSCN1 depletion significantly reduced organoid size and growth rate, while FSCN1 overexpression enhanced organoid expansion and Ki-67 positivity (Fig. S5F–G). Transwell assays demonstrated that silencing FSCN1 reduced cell migration and invasion, while FSCN1 overexpression enhanced these properties (Fig. S5H–I). Flow-cytometric analysis revealed that FSCN1 silencing increased apoptosis, whereas FSCN1 overexpression conferred apoptosis resistance (Fig. S5J–K). These results indicate that FSCN1 phenocopies the tumor-promoting roles of La/SSB in vitro. In line with the in vitro results, xenograft studies showed that FSCN1 depletion significantly decreased tumor size and weight, whereas FSCN1 overexpression promoted tumor growth (Fig. S6A–D, S6I–L). Immunohistochemical staining of xenograft tissues confirmed that FSCN1-deficient tumors exhibited decreased Ki-67 and increased cleaved caspase-3 levels, while FSCN1-overexpressing tumors showed the opposite pattern (Fig. S6E–H, S6M–P). Together, these data establish that FSCN1 recapitulates La/SSB-driven oncogenic behavior both in vitro and in vivo. To further determine whether FSCN1 is essential for La/SSB-induced tumorigenesis, rescue experiments were performed by re-expressing FSCN1 in La/SSB-knockdown FaDu cells (Fig. 5A–B). Restoration of FSCN1 expression significantly rescued the proliferative, migratory, and invasive deficiencies induced by La/SSB loss, as shown by CCK-8, colony-formation, EdU, and Transwell assays (Fig. 5C–E, 5G). The increase in apoptosis caused by La/SSB depletion was also reversed upon FSCN1 re-expression (Fig. 5F). In vivo, FSCN1 re-expression in La/SSB-deficient FaDu cells restored xenograft tumor growth and lung-metastatic colonization, accompanied by elevated Ki-67 and reduced cleaved caspase-3 expression (Fig. 5H–N). Collectively, these results demonstrate that FSCN1 not only phenocopies but also mediates the tumor-promoting functions of La/SSB, serving as a key downstream effector in HNSCC progression. 6. TFAP2C directly activates FSCN1 transcription downstream of La/SSB To delineate the transcriptional mechanism linking La/SSB to FSCN1 , we performed integrated transcription-factor prediction and correlation analyses. Among candidate regulators identified from JASPAR and ChIP-Atlas databases, in the TCGA-HNSC cohort, TFAP2C exhibited the highest positive correlation with FSCN1 expression (r = 0.214, P < 0.001). (Fig. 6A–C). TFAP2C transcript and protein levels were significantly elevated in HNSCC tissues compared with adjacent mucosa, and higher expression was associated with advanced clinical stage and reduced overall survival (Fig. 6D–E). To test whether TFAP2C directly regulates FSCN1 , we modulated TFAP2C expression in HNSCC cells. Silencing TFAP2C in FaDu cells markedly decreased FSCN1 mRNA and protein, whereas TFAP2C overexpression in TU177 cells induced robust FSCN1 up-regulation (Fig. 6F). In PDOs, TFAP2C depletion suppressed organoid growth and proliferation, while TFAP2C overexpression enhanced these features, mirroring La/SSB-dependent phenotypes (Fig. 6G–H). Bioinformatic motif analysis revealed a high-affinity TFAP2C-binding site within the FSCN1 promoter region (Fig. 6I). Chromatin immunoprecipitation (ChIP) assays confirmed TFAP2C enrichment at this site (Fig. 6J–K). Dual-luciferase reporter assays further demonstrated that TFAP2C significantly increased FSCN1 promoter activity, In contrast, mutation of the binding site eliminated this effect (Fig. 6L). Importantly, La/SSB knockdown diminished TFAP2C occupancy at the FSCN1 promoter, whereas La/SSB overexpression enhanced it (Fig. 6M). Together, these data indicate that La/SSB promotes FSCN1 transcription by facilitating TFAP2C recruitment to its promoter, forming a transcriptional activation axis critical for HNSCC progression. 7. Inhibition or loss of La/SSB enhances cisplatin sensitivity in PDOs and La/ssb cKO mice Given that La/SSB promotes tumor survival, we next investigated whether its inhibition could potentiate the efficacy of CDDP, a first-line chemotherapy for HNSCC. In patient-derived organoids, La/SSB knockdown significantly sensitized tumors to CDDP treatment, producing a synergistic suppression of viability compared with either intervention alone (Fig. 7A–B). Immunofluorescence staining showed pronounced decreases in Ki-67-positive cells and increases in cleaved-caspase-3-positive cells in the combination group, indicating enhanced apoptosis (Fig. 7C–D). To evaluate the in vivo therapeutic significance, conditional La/ssb cKO mice were produced by crossing La/ssb fl/fl mice with Krt14-Cre/ERT2 transgenic mice, followed by induction with tamoxifen. (Fig. 7E). After 4-NQO-induced oral carcinogenesis, La/ssb cKO mice developed markedly fewer and smaller tongue tumors compared with wild-type controls (Fig. 7F). Combination therapy with CDDP further suppressed tumor burden and invasiveness (Fig. 7G). Histologic analysis revealed extensive necrosis and reduced Ki-67 expression in tumors from La/ssb cKO + CDDP mice (Fig. 7H). These findings demonstrate that La/SSB loss profoundly enhances the antitumor efficacy of cisplatin, suggesting that pharmacologic La/SSB inhibition could be exploited to overcome platinum resistance in HNSCC. 8. Coordinated activation of the La/SSB–TFAP2C–FSCN1 axis correlates with malignant progression and poor prognosis To establish the clinical relevance of this regulatory cascade, we analyzed La/SSB, TFAP2C, and FSCN1 expression in 60 paired HNSCC and adjacent normal samples. IHC analysis revealed a strong positive correlation among the three proteins (Fig. 8A), and tumors exhibiting co-upregulation of all three markers displayed significantly higher T stage and lymph-node metastasis rates. ROC curve analysis demonstrated that these genes exhibited high diagnostic performance for differentiating malignant from normal tissues, with AUCs of 0.872 for La/SSB, 0.868 for TFAP2C, and 0.862 for FSCN1 (Fig. 8B). Consistent trends were observed in the TCGA-HNSC cohort (AUC = 0.844, 0.619, 0.948, respectively; Fig. 8C). Representative IHC staining demonstrated that tumor regions with intense La/SSB expression exhibited concurrent nuclear TFAP2C accumulation and elevated cytoplasmic FSCN1 levels (Fig. 8D). Quantitative assessment showed that patients with elevated levels of all three markers exhibited significantly worse overall survival compared with individuals with low or intermediate expression (Fig. 8E). Collectively, these results define a clinically relevant La/SSB–TFAP2C–FSCN1 axis whose coordinated activation drives HNSCC aggressiveness and predicts adverse patient outcomes. Discussion The present study identifies La/SSB as a pivotal oncogenic driver in HNSCC. Through multi-omics integration and multi-model validation, we reveal that La/SSB reprograms tumor transcriptional activity by enhancing TFAP2C-dependent activation of FSCN1 , thereby coupling RNA-binding dynamics with chromatin-based gene regulation. This work expands the functional paradigm of RNA-binding proteins (RBPs) beyond their canonical post-transcriptional roles, positioning La/SSB as a molecular bridge that links RNA metabolism, cytoskeletal remodeling, and chemotherapeutic resistance in HNSCC. La/SSB was originally described as an autoimmune antigen in Sjögren syndrome, but accumulating evidence indicates that its dysregulation contributes to tumorigenesis[8, 9, 20-22]. Increased La/SSB expression has been observed in hepatocellular carcinoma, breast cancer, and non-small-cell lung cancer, where it stabilizes oncogenic mRNAs and sustains translation under stress[11, 23-25]. Our findings extend this oncogenic landscape to HNSCC, showing that La/SSB upregulation correlates with advanced disease and poor prognosis. Importantly, its loss attenuates tumor growth and metastatic potential across both in vitro and in vivo systems, underscoring its functional indispensability for HNSCC progression. These data highlight La/SSB as a member of an emerging class of RBPs that exert transcription-like control through selective modulation of chromatin-associated complexes. Mechanistically, our integrative RNA-seq and ATAC-seq analyses pinpoint FSCN1 as the dominant downstream effector of La/SSB. FSCN1 encodes an actin-bundling protein essential for the formation of filopodia and invadopodia, cellular structures that enable motility and invasion[26-29]. In normal epithelia, FSCN1 expression is tightly suppressed, whereas its reactivation is a hallmark of metastatic carcinomas[30, 31]. The finding that La/SSB silencing diminishes FSCN1 promoter accessibility, while FSCN1 restoration rescues La/SSB loss-of-function phenotypes, establishes a direct mechanistic dependency. This epistatic relationship reveals a hierarchical control in which La/SSB governs cytoskeletal plasticity through transcriptional remodeling rather than mere mRNA stabilization. Upstream of FSCN1 , we identified TFAP2C as a transcriptional intermediary that translates La/SSB activity into promoter-specific gene activation. TFAP2C is a context-dependent transcription factor known to remodel chromatin accessibility and coordinate oncogenic programs such as epithelial–mesenchymal transition and therapy resistance[32-35]. Prior studies have shown that TFAP2C cooperates with lineage-specific factors like p63 or GATA3 to shape enhancer landscapes in epithelial tumors[36-38]. Our data now suggest that La/SSB may facilitate TFAP2C recruitment to target promoters, potentially by modulating chromatin architecture or RNA–protein scaffold formation. This crosstalk exemplifies a broader principle whereby RBPs indirectly dictate transcriptional outcomes by influencing transcription factor loading and nucleosome accessibility—a conceptual convergence of post-transcriptional and epigenetic regulation. Functionally, La/SSB also confers protection against genotoxic stress, contributing to CDDP resistance. Platinum compounds induce DNA crosslinks and activate DNA-damage response pathways, leading to apoptosis in sensitive cells. The survival advantage conferred by La/SSB likely stems from its ability to sustain translation of DNA-repair and anti-apoptotic transcripts during stress adaptation. Inhibition or deletion of La/SSB markedly enhanced CDDP efficacy in organoid and conditional-knockout models, suggesting that targeting La/SSB may disrupt this adaptive translational network. These findings align with prior reports that RBPs such as IGF2BP2 or YBX1 mediate similar resistance programs, reinforcing the idea that translational plasticity constitutes a major determinant of chemoresistance in epithelial malignancies[39]. Conceptually, the La/SSB–TFAP2C–FSCN1 axis delineated here underscores a multilayer regulatory circuit in which an RNA-binding protein indirectly orchestrates transcriptional activation to promote cytoskeletal reorganization, invasion, and therapeutic evasion. This dual control over RNA fate and chromatin accessibility may represent a general strategy exploited by tumors to sustain malignant phenotypes under environmental and therapeutic stress. From a translational perspective, La/SSB’s tumor-restricted expression and multifunctional role render it an attractive candidate for therapeutic intervention. Pharmacologic or RNA-based La/SSB inhibition, particularly when combined with platinum-based chemotherapy, could enhance treatment responses in aggressive HNSCC. Furthermore, La/SSB and FSCN1 expression signatures may serve as biomarkers for risk stratification and therapeutic guidance. In conclusion, this study uncovers a noncanonical regulatory axis centered on La/SSB that integrates RNA-binding and chromatin-modulating functions to drive HNSCC progression and chemoresistance. These findings broaden our understanding of how RBPs rewire oncogenic signaling and open new avenues for therapeutic targeting of RBP-dependent vulnerabilities in epithelial cancers. Abbreviations HNSC : Head and Neck Squamous Cell Carcinoma ANM : adjacent non-malignant tissues La/SSB : Small RNA binding exonuclease protection factor La FSCN1 : Fascin actin-bundling protein 1 TFAP2C : Transcription factor activating protein 2 gamma CDDP : Cisplatin DMSO : Dimethyl Sulfoxide PBS : phosphate-buffered saline HEK 293T : human embryonic kidney 293T qRT-PCR : quantitative real-time PCR CCK-8 : Cell Counting Kit-8 PDO : patient-derived Oragnoid CDX : cell line-derived xenograft model NOK : Normal oral epithelial cell line ATCC : American Type Culture Collection DEGs : Differentially expressed genes DARs : Differentially accessible regions 4NQO : 4-nitroquinoline N-oxide ATAC : Assay for Transposase Accessible Chromatin IGV : Integrative Genomics Viewer IF : immunofluorescence ChIP : chromatin immunoprecipitation H&E : Hematoxylin and eosin IHC : immunohistochemistry siRNAs : small interfering RNA shRNA : short hairpin RNA RNA-seq : RNA sequencing TCGA : The Cancer Genome Atlas CoIP : Co-immunoprecipitation OS : Overall survival TFs : Transcription factors ROC : Receiver Operating Characteristic AUC : Area Under the Curve Declarations Availability of data and materials The data supporting the findings of this study can be accessed from the GEO repository under accession numbers GSE283322 and GSE283323. Acknowledgments This work was supported by the Natural Science Foundation of China (82171127, 82371133, 82171128 and 82303021), Anhui Provincial Natural Science Foundation (2208085MH239), the Natural Science Foundation of Universities of Anhui Province (2022AH051134), Discipline Construction Project of the First Affiliated Hospital of Anhui Medical University (NO. 4245) and Anhui Medical University Foundation（2023xkj146). Author contributions SYM XJZ and YFH supervised the project; SXL, SYM, XJZ and YFH designed the experiments; SXL, WTZ and WWL performed the experiments; SXL, WTZ, DSC, JP, HL, JJZ, XMW, MJZ, MJW, CQC, SCL, XLY, QX and GYH analyzed the data; SXL, XJZ and DSC wrote the paper; SYM and YFH revised the paper. All authors read and approved the manuscript. Declaration of interests The authors declare no competing interests. Clinical trial registration: Clinical trial number: not applicable. 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Univariate and multivariate Cox regression analyses of the correlation between clinical characteristics with overall survival Univariate analysis Multivariate analysis Characteristics Total(N) Hazard ratio (95% CI) P value Hazard ratio (95% CI) P value Age 478 60 241 1.088 (0.770 - 1.538) 0.632 1.134 (0.794 - 1.619) 0.489 Gender 478 Female 123 Reference Reference Male 355 0.968 (0.652 - 1.439) 0.874 0.942 (0.628 - 1.415) 0.775 Histologic grade 463 G1&G2 347 Reference Reference G3&G4 116 1.055 (0.715 - 1.557) 0.788 0.986 (0.666 - 1.460) 0.944 Clinical stage 464 Stage I&Stage II 107 Reference Reference Stage III&Stage IV 357 1.161 (0.759 - 1.775) 0.492 1.091 (0.705 - 1.687) 0.695 SSB 478 Low 239 Reference Reference High 239 1.812 (1.270 - 2.587) 0.001 1.924 (1.337 - 2.771) < 0.001 The bold values are defined as significant (p < 0.05). Table 2. Univariate and multivariate Cox regression analyses of the correlation among the clinical characteristics with disease-specific survival Univariate analysis Multivariate analysis Characteristics Total(N) Hazard ratio (95% CI) P value Hazard ratio (95% CI) P value Age 503 60 256 1.262 (0.964 - 1.653) 0.090 1.349 (1.015 - 1.791) 0.039 Gender 503 Female 134 Reference Reference Male 369 0.760 (0.571 - 1.012) 0.061 0.772 (0.571 - 1.043) 0.092 Histologic grade 483 G1&G2 362 Reference Reference G3&G4 121 0.942 (0.690 - 1.286) 0.706 0.894 (0.653 - 1.225) 0.487 Clinical stage 489 Stage I&Stage II 114 Reference Reference Stage III&Stage IV 375 1.226 (0.885 - 1.700) 0.221 1.191 (0.849 - 1.671) 0.312 SSB 503 Low 251 Reference Reference High 252 1.563 (1.193 - 2.049) 0.001 1.717 (1.296 - 2.276) < 0.001 The bold values are defined as significant (p < 0.05). Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementaryTableS1.xlsx Supplementary Table S1 SupplementaryTableS2.xlsx Supplementary Table S2 DocumentS1.SupplementaryFigureS1S7TableS3S11.docx Document S1.Supplementary Figure S1-S7, Table S3- S11 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 11 Feb, 2026 Review # 3 received at journal 09 Feb, 2026 Review # 1 received at journal 02 Feb, 2026 Review # 2 received at journal 26 Jan, 2026 Reviewer # 3 agreed at journal 22 Jan, 2026 Reviewer # 2 agreed at journal 15 Jan, 2026 Reviewer # 1 agreed at journal 15 Jan, 2026 Reviewers invited by journal 15 Jan, 2026 Submission checks completed at journal 04 Jan, 2026 Editor assigned by journal 18 Dec, 2025 First submitted to journal 18 Dec, 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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1","display":"","copyAsset":false,"role":"figure","size":16098290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAs a new diagnostic biomarker, La/SSB is highly expressed in HNSC and correlates with poorer prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eC\u003c/strong\u003e) La/SSB expression in ANM and tumor tissues was analyzed using the TCGA-HNSC (\u003cstrong\u003eA\u003c/strong\u003e), GSE127165 (\u003cstrong\u003eB\u003c/strong\u003e), and GSE178537 (\u003cstrong\u003eC\u003c/strong\u003e) datasets. Data are presented as mean ±SD; ***P \u0026lt; 0.001. (\u003cstrong\u003eD\u003c/strong\u003e-\u003cstrong\u003eE\u003c/strong\u003e) Kaplan–Meier curves depicting overall survival (\u003cstrong\u003eD\u003c/strong\u003e) and disease-specific survival (\u003cstrong\u003eE\u003c/strong\u003e) of HNSC patients from the TCGA cohort stratified by La/SSB expression. (\u003cstrong\u003eF\u003c/strong\u003e) Western blot analysis of paired ANM and HNSC tissues (n = 12). (\u003cstrong\u003eG\u003c/strong\u003e-\u003cstrong\u003eH\u003c/strong\u003e) H-score quantification of immunohistochemical staining (\u003cstrong\u003eG\u003c/strong\u003e) with representative images shown in (\u003cstrong\u003eH\u003c/strong\u003e). Scale bars: 200 μm (low magnification), 50 μm (high magnification). Data are mean ±SD; *P \u0026lt; 0.05, ***P \u0026lt; 0.001. (\u003cstrong\u003eI\u003c/strong\u003e) Kaplan–Meier analysis of overall survival in 60 HNSC patients based on median H-score of La/SSB IHC staining, dividing patients into high (n = 30) and low (n = 30) expression groups. (\u003cstrong\u003eJ\u003c/strong\u003e) La/SSB mRNA and protein levels in NOK and HNSC cell lines (TU177, FaDu, TU686, LIU-LSC-1, TU212) were assessed by qRT-PCR and western blot. Data are mean ± SD; **P \u0026lt; 0.01. (\u003cstrong\u003eK\u003c/strong\u003e) Representative immunofluorescence images of La/SSB in NOK and HNSC cell lines. Scale bar: 20 μm.\u003c/p\u003e","description":"","filename":"XXX1.png","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/8d2d1a439cb44fb594fb14c6.png"},{"id":100749454,"identity":"fb85eb2f-aa02-45e8-abe4-9d10d9b60c35","added_by":"auto","created_at":"2026-01-21 04:21:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29036969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLa/SSB enhances tumorigenic capacity of HNSC cells in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA \u003c/strong\u003eand\u003cstrong\u003e B\u003c/strong\u003e) FaDu cells were transduced with lentiviruses expressing shRNAs targeting La/SSB (shLa/SSB#1 and shLa/SSB#2) or a scrambled control sequence (shSc) (\u003cstrong\u003eA\u003c/strong\u003e), whereas TU177 cells were transduced with either a control vector or lentiviruses encoding La/SSB (\u003cstrong\u003eB\u003c/strong\u003e); La/SSB protein levels were assessed by western blotting. (\u003cstrong\u003eC\u003c/strong\u003e–\u003cstrong\u003eD\u003c/strong\u003e) Cell proliferation was evaluated via EdU incorporation assays, with scale bars of 100 μm. (\u003cstrong\u003eE\u003c/strong\u003e) Schematic representation illustrates the enzymatic digestion and initial culture procedure for HNSC organoids. (\u003cstrong\u003eF\u003c/strong\u003e and\u003cstrong\u003e I\u003c/strong\u003e) Representative immunohistochemical images of CK13, p63, and La/SSB staining in HNSC tissues and organoids are shown, with scale bars of 40 μm for tissues and 100 μm for organoids. (\u003cstrong\u003eF \u003c/strong\u003eand\u003cstrong\u003e G\u003c/strong\u003e) La/SSB protein expression in organoids was confirmed using western blotting. (\u003cstrong\u003eH \u003c/strong\u003eand\u003cstrong\u003e I\u003c/strong\u003e) Representative images display organoid diameters and Ki-67 immunofluorescence intensity following La/SSB knockdown or overexpression, with IHC scale bars of 400 μm (low magnification) and 100 μm (high magnification) and IF scale bars of 40 μm; organoid diameter quantification (n = 30) is presented as mean ± SD (**P \u0026lt; 0.01, ****P \u0026lt; 0.0001). (\u003cstrong\u003eJ \u003c/strong\u003eand\u003cstrong\u003e K\u003c/strong\u003e) Transwell assays were performed to assess migration and invasion in La/SSB-knockdown FaDu cells and La/SSB-overexpressing TU177 cells, with scale bars of 100 μm; quantitative data are shown as mean ± SD (**P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"XXX2.png","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/91d7609952608bb321f7f275.png"},{"id":100749434,"identity":"12944dd2-facc-48ea-a623-1759d49a1737","added_by":"auto","created_at":"2026-01-21 04:21:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27755037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLa/SSB enhances tumorigenic capacity of HNSC cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e–\u003cstrong\u003eH\u003c/strong\u003e) FaDu cells with the indicated modifications were subcutaneously injected into mice to monitor tumor growth. Representative images of the tumors are shown (\u003cstrong\u003eA\u003c/strong\u003e), along with tumor weights (\u003cstrong\u003eB\u003c/strong\u003e), tumor volumes (\u003cstrong\u003eC\u003c/strong\u003e), and body weights of the mice over time (\u003cstrong\u003eD\u003c/strong\u003e). Immunohistochemical staining of xenograft tissues for La/SSB, Ki-67, and cleaved caspase-3 is presented in (\u003cstrong\u003eE\u003c/strong\u003e) with a scale bar of 50 μm, and the corresponding quantitative analyses are shown in (\u003cstrong\u003eF\u003c/strong\u003e–\u003cstrong\u003eH\u003c/strong\u003e). Data are expressed as mean ± SD (**P \u0026lt; 0.01, ****P \u0026lt; 0.0001). (\u003cstrong\u003eI \u003c/strong\u003eand\u003cstrong\u003eJ\u003c/strong\u003e) Representative images of lungs (upper panels, scale bar 1 cm) and H\u0026amp;E-stained lung sections (lower panels, scale bar 2 mm) are shown, with red arrowheads indicating metastatic nodules. (\u003cstrong\u003eK \u003c/strong\u003eand\u003cstrong\u003e L\u003c/strong\u003e) Quantification of lung metastatic nodules and lung weights from all animals is presented, with data shown as mean ± SD (n = 5 mice per group; *P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"XXX3.png","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/a97981501fe186082ceda16e.png"},{"id":100749430,"identity":"c470561f-46d3-458a-b903-f49fb932cd73","added_by":"auto","created_at":"2026-01-21 04:21:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6255166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFSCN1 is a key functional downstream target of La/SSB.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA \u003c/strong\u003eand\u003cstrong\u003e B\u003c/strong\u003e) shLa/SSB#1 LIU-LSC-1 cells and corresponding control cells were subjected to RNA-seq and ATAC-seq analyses. A volcano plot depicts differentially expressed genes (|log₂FC| \u0026gt; 1, p \u0026lt; 0.05) (\u003cstrong\u003eA\u003c/strong\u003e), while heatmaps and enrichment plots illustrate normalized ATAC-seq signal densities after La/SSB knockdown (\u003cstrong\u003eB\u003c/strong\u003e), with tracks centered at transcription start sites (TSS) and extending ±2 kb. (\u003cstrong\u003eC\u003c/strong\u003e) A schematic workflow shows the screening process for downstream targets of La/SSB. A Venn diagram identifies seven overlapping genes from two datasets: Dataset 1 includes downregulated genes from RNA-seq and ATAC-seq (log₂FC \u0026lt; −1, p \u0026lt; 0.05), and Dataset 2 comprises upregulated genes from the TCGA-HNSC dataset (log₂FC \u0026gt; 2, p \u0026lt; 0.01). These seven genes were further filtered based on their expression correlating with poorer patient prognosis in the TCGA-HNSC dataset (p \u0026lt; 0.01). (\u003cstrong\u003eD\u003c/strong\u003e–\u003cstrong\u003eF\u003c/strong\u003e) FSCN1 expression in HNSC and adjacent normal mucosa was analyzed using TCGA (\u003cstrong\u003eD\u003c/strong\u003e), GSE127165 (\u003cstrong\u003eE\u003c/strong\u003e), and GSE178537 (\u003cstrong\u003eF\u003c/strong\u003e) datasets. (\u003cstrong\u003eG\u003c/strong\u003e) Kaplan–Meier analysis of overall survival in HNSC patients stratified by median FSCN1 expression (high, n = 252; low, n = 251) is presented. (\u003cstrong\u003eH\u003c/strong\u003e) Visualization of the FSCN1 locus with ATAC-seq signals was performed using the Integrative Genomics Viewer (IGV). (\u003cstrong\u003eI \u003c/strong\u003eand\u003cstrong\u003e J\u003c/strong\u003e) In La/SSB knockdown FaDu cells and La/SSB-overexpressing TU177 cells, FSCN1 expression was assessed by qRT-PCR and western blotting compared with respective controls. (\u003cstrong\u003eK\u003c/strong\u003e) H-score quantification of FSCN1 immunostaining is shown, with data expressed as mean ± SD (*P \u0026lt; 0.05, ***P \u0026lt; 0.001). (\u003cstrong\u003eL\u003c/strong\u003e) Kaplan–Meier analysis of overall survival in 60 HNSC patients based on FSCN1 protein levels is presented; patients were divided into high (n = 30) and low (n = 30) groups according to the median H-score of IHC staining.\u003c/p\u003e","description":"","filename":"XXX4.png","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/346feacae5ae430e5285c3f5.png"},{"id":100796489,"identity":"0cc83c22-d3ab-4d81-b476-cf5cd342ce7c","added_by":"auto","created_at":"2026-01-21 13:43:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31922452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFSCN1 is a functionally important downstream target of La/SSB in HNSC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA \u003c/strong\u003eand\u003cstrong\u003e B\u003c/strong\u003e) shLa/SSB#1 FaDu cells and corresponding control cells were transduced with lentiviruses carrying either human FSCN1 cDNA or an empty vector. FSCN1 expression levels were evaluated by western blotting (\u003cstrong\u003eA\u003c/strong\u003e) and qRT-PCR (\u003cstrong\u003eB\u003c/strong\u003e). (\u003cstrong\u003eC\u003c/strong\u003e–\u003cstrong\u003eE\u003c/strong\u003e) Cell proliferation was assessed using colony formation (\u003cstrong\u003eC\u003c/strong\u003e) and EdU incorporation assays (\u003cstrong\u003eD\u003c/strong\u003e), with scale bars of 100 μm; data are presented as mean ± SD (**P \u0026lt; 0.01, ****P \u0026lt; 0.0001). (\u003cstrong\u003eE\u003c/strong\u003e) Representative images of organoid diameters are shown, with scale bars of 400 μm (low magnification) and 100 μm (high magnification); tumor organoid diameter quantification is based on n = 30 organoids. (\u003cstrong\u003eF\u003c/strong\u003e) Apoptosis was analyzed by flow cytometry, with results expressed as mean ± SD (***P \u0026lt; 0.001). (\u003cstrong\u003eG\u003c/strong\u003e) Transwell assays were used to determine migration and invasion capacities, with scale bars of 100 μm; quantitative data are shown as mean ± SD (****P \u0026lt; 0.0001). (\u003cstrong\u003eH\u003c/strong\u003e–\u003cstrong\u003eM\u003c/strong\u003e) The indicated FaDu cells were subcutaneously injected into mice to monitor tumor growth. Representative tumor images (\u003cstrong\u003eH\u003c/strong\u003e), tumor weights (\u003cstrong\u003eI\u003c/strong\u003e), tumor volumes (\u003cstrong\u003eJ\u003c/strong\u003e), and body weights (\u003cstrong\u003eK\u003c/strong\u003e) are presented. Immunohistochemical staining of xenograft tissues for La/SSB, Ki-67, and cleaved caspase-3 is shown in (\u003cstrong\u003eL\u003c/strong\u003e) with scale bars of 50 μm, and quantitative analyses for La/SSB, FSCN1, Ki-67, and cleaved caspase-3 are presented in (M) (mean ± SD, n = 5 mice per group; ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001). (\u003cstrong\u003eN\u003c/strong\u003e) Representative images of lungs (scale bar 1 cm) and H\u0026amp;E-stained sections (scale bar 1 mm) illustrate metastatic lesions after tail vein injection; red arrowheads indicate metastatic nodules. Quantification of lung metastatic nodules and lung weights is summarized, with data shown as mean ± SD (n = 5 mice per group; *P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"XXX5.png","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/9303bccb1a377e2421fd0c3f.png"},{"id":100796607,"identity":"3309181d-ad54-4360-a77b-bfb448588307","added_by":"auto","created_at":"2026-01-21 13:44:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":14823654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTFAP2C transcriptionally activates La/SSB gene expression in HNSC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Venn diagram showing transcription factors positively associated with La/SSB expression in the TCGA-HNSC cohort. (\u003cstrong\u003eB\u003c/strong\u003e) Comparative analysis of TFAP2C and SP1 expression between ANM and HNSC tumor tissues based on TCGA-HNSC data. (\u003cstrong\u003eC\u003c/strong\u003e) Pearson correlation analyses of FSCN1 with TFAP2C and SP1 in the TCGA dataset. (\u003cstrong\u003eD\u003c/strong\u003e) H-score quantification of immunohistochemical staining with representative images; scale bar, 40 μm. Data are shown as mean ± SD. *P \u0026lt; 0.05, ***P \u0026lt; 0.001. (\u003cstrong\u003eE\u003c/strong\u003e) Kaplan–Meier survival analysis of 60 HNSC patients stratified by median TFAP2C protein expression, with high (n = 30) and low (n = 30) groups. (\u003cstrong\u003eF\u003c/strong\u003e) FaDu cells were transfected with TFAP2C-targeting shRNAs or control shRNA (shSc). TFAP2C protein and mRNA levels were measured by western blotting and qRT-PCR. FSCN1 expression in TU177 cells was also evaluated using the same assays. Data are presented as mean ± SD; ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. (\u003cstrong\u003eG\u003c/strong\u003e and\u003cstrong\u003eH\u003c/strong\u003e) Representative images of tumor organoid diameters following TFAP2C knockdown or overexpression, with scale bars of 400 μm (low magnification) and 100 μm (high magnification); n = 30 organoids. (\u003cstrong\u003eI\u003c/strong\u003e) Analysis of TFAP2C-binding peaks upstream of the FSCN1 promoter using ChIP-Atlas. (\u003cstrong\u003eJ\u003c/strong\u003e) Schematic diagram depicting the predicted TFAP2C binding site within the La/SSB promoter region. (\u003cstrong\u003eK\u003c/strong\u003e) ChIP-PCR assay assessing TFAP2C occupancy at the FSCN1 promoter in TU177 cells. (\u003cstrong\u003eL\u003c/strong\u003e) 293T cells were co-transfected with TFAP2C-Luc or FSCN1-mut constructs along with TFAP2C-pcDNA3.1 or empty vector; luciferase activity was measured 24 h post-transfection. Data are mean ± SD; ****P \u0026lt; 0.0001. (\u003cstrong\u003eM\u003c/strong\u003e) ChIP assays in vector control and TFAP2C-overexpressing TU177 cells using FSCN1 antibodies or control IgG, with qPCR amplification of regions flanking the predicted TFAP2C binding site. Results are expressed as the percentage of input DNA. Data are mean ± SD; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"XXX6.png","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/be48a548eaa8d8ce5a609132.png"},{"id":100749438,"identity":"d0be6bfb-5bfe-48a6-98c4-6b23cc486fea","added_by":"auto","created_at":"2026-01-21 04:21:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":34158857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting La/SSB promotes the CDDP treatment effect in PDO and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Cell viability of PDOs treated with shLa/SSB and/or CDDP. Organoids were imaged at low (100 μm) and high (20 μm) magnifications. Viability was assessed using the Cell-Titer Glo-3D assay. Data are presented as mean ± SD; ****P \u0026lt; 0.0001. (\u003cstrong\u003eB\u003c/strong\u003e) Representative immunofluorescence (IF) images showing La/SSB and Ki-67 expression. Scale bar, 20 μm. Quantification of Ki-67-positive cells per organoid (n = 5) is shown as mean ± SD; *P \u0026lt; 0.05, **P \u0026lt; 0.01. (\u003cstrong\u003eC\u003c/strong\u003e) Representative IF images for La/SSB and Cleaved caspase-3 in PDOs. Scale bar, 20 μm. The proportion of Cleaved caspase-3-positive cells per organoid (n = 5) is quantified. Data are mean ± SD; ****P \u0026lt; 0.0001. (\u003cstrong\u003eD\u003c/strong\u003e) Schematic of the experimental design for La/ssbcKO mice. La/ssbcKO mice were generated by crossing La/ssb^fl/fl mice with Krt14Cre/ERT2 mice. After 16 weeks of 4NQO-induced tongue injury, tamoxifen was administered intraperitoneally to induce conditional knockout, followed by four weeks of CDDP treatment prior to euthanasia. (\u003cstrong\u003eE\u003c/strong\u003e) Representative images of visible tongue lesions and quantification of lesion areas across treatment groups. Scale bar, 1 mm. Data are mean ± SD; ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. (\u003cstrong\u003eF\u003c/strong\u003e) H\u0026amp;E-stained sections illustrating lesion histology and quantification of invasion grades. Scale bars: 100 μm (low magnification), 50 μm (high magnification). Error bars represent mean ± SD; ***P \u0026lt; 0.001. (\u003cstrong\u003eG\u003c/strong\u003e) IF images showing La/SSB (red) and Ki-67 (green) in tongue lesions, with DAPI-stained nuclei (blue). The percentage of Ki-67-positive cells per lesion is shown. Scale bar, 20 μm. Data are mean ± SD; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. (\u003cstrong\u003eH\u003c/strong\u003e) Representative IF images of La/SSB (green) and Cleaved caspase-3 (red) in lesions. Scale bar, 20 μm. Quantification of Cleaved caspase-3-positive cells per organoid (n = 5) is presented as mean ± SD; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"XXX7.png","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/2029fe04fae7d6972bfb3202.png"},{"id":100749439,"identity":"c6a52555-c6a7-412b-9167-050b6644cc0e","added_by":"auto","created_at":"2026-01-21 04:21:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":18522185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of the La/SSB mediated TFAP2C-FSCN1 signaling pathway is associated with malignant progression and poor prognosis of HNSC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Correlation analysis among La/SSB, TFAP2C, and FSCN1 expression levels in 60 HNSC tissue samples. Spearman’s correlation coefficients are indicated in the upper-right corners. Scatterplot matrices with fitted regression lines for the respective gene pairs are shown in the lower-left panels. Data are presented as mean ± SD; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. (\u003cstrong\u003eB \u003c/strong\u003eand\u003cstrong\u003e C\u003c/strong\u003e) Receiver operating characteristic (ROC) curves assessing the diagnostic performance of La/SSB, TFAP2C, and FSCN1 expression based on H-scores from 60 HNSC patient tissues (\u003cstrong\u003eB\u003c/strong\u003e) and TCGA cohort data (\u003cstrong\u003eC\u003c/strong\u003e). (\u003cstrong\u003eD\u003c/strong\u003e) Representative immunohistochemical (IHC) images of La/SSB, TFAP2C, and FSCN1 in HNSC tissues. Scale bar, 100 μm. (\u003cstrong\u003eE\u003c/strong\u003e) Schematic model depicting the oncogenic role of La/SSB-activated TFAP2C-FSCN1 signaling in HNSC, highlighting its effects on promoting cell proliferation and migration, inhibiting apoptosis, and contributing to chemoresistance.\u003c/p\u003e","description":"","filename":"XXX8.png","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/21bb0a02831ceeb75508385a.png"},{"id":100749442,"identity":"60b320db-27ab-49df-af39-e4eb1d680ec1","added_by":"auto","created_at":"2026-01-21 04:21:43","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4581333,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S1\u003c/p\u003e","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/e06a460de105f09133b6195a.xlsx"},{"id":100857711,"identity":"36580ee5-dee5-461c-a1f1-1638efb71818","added_by":"auto","created_at":"2026-01-22 07:20:56","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1676792,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S2\u003c/p\u003e","description":"","filename":"SupplementaryTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/65a9f02c238dede845d0cd04.xlsx"},{"id":100749421,"identity":"ab6f2d41-1bd1-4699-857c-b12c0300d7d6","added_by":"auto","created_at":"2026-01-21 04:21:41","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4545853,"visible":true,"origin":"","legend":"Document S1.Supplementary Figure S1-S7, Table S3- S11","description":"","filename":"DocumentS1.SupplementaryFigureS1S7TableS3S11.docx","url":"https://assets-eu.researchsquare.com/files/rs-8395850/v1/af58f135b5f4197c58842a00.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"The RNA-binding protein La/SSB drives head and neck squamous cell carcinoma progression through TFAP2C-mediated transcriptional activation of FSCN1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHead and neck squamous cell carcinoma (HNSCC) represents the most prevalent histological subtype of head and neck cancers, accounting for more than 90% of cases[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Notwithstanding ongoing improvements in surgical approaches, radiotherapy, and the addition of immunotherapy, the five-year overall survival rate remains below 65%[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Local recurrence, distant metastasis, and resistance to chemotherapy\u0026mdash;particularly to platinum-based regimens\u0026mdash;remain major obstacles to successful treatment[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The molecular mechanisms underlying HNSCC progression and therapy resistance therefore warrant in-depth investigation to identify new prognostic markers and therapeutic targets.\u003c/p\u003e \u003cp\u003eThe La antigen, also known as Sj\u0026ouml;gren syndrome type B antigen (La/SSB or LARP3), is an evolutionarily conserved RNA-binding protein that participates in RNA maturation, stability, and translation[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Beyond its physiological roles in RNA metabolism, aberrant overexpression of La/SSB has been reported in multiple malignancies, including hepatocellular carcinoma, lung cancer, and breast cancer[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Elevated La/SSB levels are often associated with enhanced tumor proliferation, invasion, and resistance to genotoxic stress, suggesting that La/SSB may function as an oncogenic factor in cancer biology[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the specific role and regulatory mechanisms of La/SSB in HNSCC remain poorly understood. Recent studies have underscored the importance of RNA-binding proteins as molecular hubs that connect post-transcriptional regulation with chromatin remodeling and signal transduction, thereby reprogramming cellular phenotypes during malignant transformation[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Whether La/SSB engages in similar regulatory cross-talk to modulate tumor progression in HNSCC has not been systematically examined.\u003c/p\u003e \u003cp\u003eIn this study, we comprehensively investigated the expression pattern, functional significance, and mechanistic basis of La/SSB in HNSCC using integrated transcriptomic and chromatin accessibility profiling. Through in vitro and in vivo validation\u0026mdash;including patient-derived organoids (PDOs), cell-derived xenografts (CDXs), and conditional La/SSB knockout mouse models\u0026mdash;we demonstrate that La/SSB exerts a central influence on tumor proliferation, invasive capacity, and resistance to chemotherapy. Collectively, these data position La/SSB as a candidate prognostic indicator and a tractable therapeutic vulnerability in HNSCC.\u003c/p\u003e"},{"header":"METHOD","content":"\u003cp\u003e\u003cstrong\u003eAnimal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHealthy BALB/c nude mice were sourced from GemPharmatech. (Jiangsu, China) and housed at the Animal Experiment Center of Anhui Medical University under conventional laboratory settings. All animal experiments were performed following protocols approved by the Ethics Committee of Anhui Medical University. (Anhui, China).\u003c/p\u003e\n\u003cp\u003eFor CDX experiments, six-week-old male BALB/c nude mice were randomly assigned to experimental groups (n = 5 per group) and subjected to different treatments. To evaluate the in vivo roles of La/SSB and FSCN1, 4 \u0026times; 10⁶ cells from the indicated cell lines were resuspended in a volume of 0.2 mL PBS and subcutaneously administered into the axilla to generate xenograft tumors. Tumor size was monitored at 3-day intervals, tumor volume was determined using the formula: volume = (length \u0026times; width\u0026sup2;)/2 (mm\u0026sup3;). At the end of the experiment, mice were euthanized, and tumors were harvested, weighed, and photographed, followed by immunohistochemical (IHC) assessment.\u003c/p\u003e\n\u003cp\u003eFor metastasis studies, nude mice received tail vein injections of 1 million genetically engineered cells suspended in 100 \u0026mu;L PBS. Eight weeks later, the mice were euthanized, and lung tissues were harvested for further analysis. Metastatic nodules were visualized under a dissecting microscope following hematoxylin and eosin (H\u0026amp;E) staining.\u003c/p\u003e\n\u003cp\u003eFor conditional knockout mice model, La/ssb\u003csup\u003efl/fl\u003c/sup\u003e mice were crossed with Krt14\u003csup\u003eCre/ERT2\u003c/sup\u003e mice to generate La/ssb\u003csup\u003efl/fl\u003c/sup\u003e; Krt14\u003csup\u003eCre/ERT2\u003c/sup\u003e offspring. All mouse strains were sourced from the Shanghai Model Organisms Center and maintained under specific pathogen-free (SPF) conditions. To establish an HNSCC model, six-week-old La/ssb\u003csup\u003ewt/wt\u003c/sup\u003e; Krt14\u003csup\u003eCre/ERT2\u003c/sup\u003e mice (designated as La/ssb\u003csup\u003eCtrl\u003c/sup\u003e) and La/ssb\u003csup\u003efl/fl\u003c/sup\u003e; Krt14\u003csup\u003eCre/ERT2\u003c/sup\u003e mice (designated as La/ssb\u003csup\u003ecKO\u003c/sup\u003e) were administered drinking water supplemented with 4-nitroquinoline 1-oxide (4NQO, 50 \u0026mu;g/mL) for 16 weeks, followed by replacement with normal drinking water to allow tumor progression. For lineage tracing and conditional deletion of La/SSB, tamoxifen was delivered via intraperitoneal injection at a dose of 120 mg/kg every other day for a total of four injections to induce Cre recombinase activity. Additionally, mice received intraperitoneal injections of cisplatin (CDDP, 5 mg/kg) once per week or saline twice per week for four consecutive weeks. Upon completion of all treatments, mice were sacrificed, and tongue tissues were harvested for downstream analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human head and neck squamous cell carcinoma (HNSC) cell line FaDu and HEK-293T cells were obtained from the American Type Culture Collection (ATCC; VA, USA). The TU686 HNSCC cell line was obtained from the BeNa Culture Collection (Beijing, China; catalogue number BNCC359450), while TU212 (catalogue number HTX2130) and human normal oral keratinocytes (NOK; catalogue number HTX2992) were supplied by Otwo Biotech (Shenzhen, China). The HNSCC cell lines TU177 and LIU-LSC-1 were characterized in previous studie[18]. All cells were maintained in RPMI 1640 medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA) and penicillin\u0026ndash;streptomycin (100 U/mL and 100 \u0026mu;g/mL, respectively; Beyotime, Jiangsu, China). Cells were cultured at 37\u0026deg;C in a humidified incubator with 5% CO₂ and routinely screened for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, #LT07-118). Additional details regarding cell sources and culture conditions are provided in \u003cstrong\u003eSupplementary Table S4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman HNSC specimens were obtained from patients treated at the First Affiliated Hospital of Anhui Medical University (Hefei, Anhui) between 2020 and 2025. Histopathological diagnoses were independently verified by a minimum of two experienced pathologists, and informed written consent was obtained from all participants before sample collection. All surgical and experimental procedures involving human tissues were performed in accordance with protocols approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University. Detailed clinicopathological characteristics, including patient age, sex, histological grade, TNM classification, lymph node status, and local invasion, are summarized in \u003cstrong\u003eSupplementary Tables S3 and S5\u003c/strong\u003e. Clinical information for patients used in the establishment of PDO models is presented in \u003cstrong\u003eSupplementary Table S6\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibodies and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetailed information regarding all antibodies utilized in this study is listed in Supplementary Table S7. CDDP was sourced from MedChemExpress (Monmouth Junction, NJ, USA). Puromycin and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (MO, USA), whereas 4NQO was obtained from Santa Cruz Biotechnology (CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol reagent (Invitrogen, CA, USA). RNA quantity and purity were assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). For cDNA synthesis, 1 \u0026mu;g of total RNA was reverse-transcribed into first-strand cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative real-time PCR (qRT-PCR) was performed on a LightCycler 96 system (Roche, Switzerland) using SYBR Premix Ex Taq II (TaKaRa, Kyoto, Japan) following the manufacturer\u0026rsquo;s protocols. Relative expression levels of target mRNAs were determined using the 2^\u0026minus;\u0026Delta;\u0026Delta;Ct method, with \u0026beta;-actin serving as the reference gene. All primers were designed and synthesized by Sangon Biotech (Shanghai, China) and are provided in \u003cstrong\u003eSupplementary Table S8\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026amp;E and immunohistochemical staining (IHC)\u003c/strong\u003e\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTumor tissues from various treatment groups or organoid models were fixed in formalin, embedded in paraffin, and sectioned for histological examination. After deparaffinization and rehydration, antigen retrieval was performed, followed by incubation with 3% hydrogen peroxide for 1 h to block endogenous peroxidase activity. Sections were then incubated with 3% bovine serum albumin for 1 h and subsequently with primary antibodies at 4 \u0026deg;C overnight. Horseradish peroxidase-conjugated secondary antibodies were applied, and immunoreactivity was visualized using DAB substrate (Beyotime), with hematoxylin serving as a nuclear counterstain. Immunohistochemical staining was semi-quantitatively evaluated using H-scores, calculated as follows: [(percentage of weak staining \u0026times; 1) + (percentage of moderate staining \u0026times; 2) + (percentage of strong staining \u0026times; 3)], resulting in scores ranging from 0 to 300.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal cellular proteins were extracted using RIPA lysis buffer (Beyotime). Protein samples were separated by 10% SDS\u0026ndash;polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk for 1 h and incubated overnight at 4 \u0026deg;C with primary antibodies (1:1000). Following washes, membranes were treated with the appropriate secondary antibodies at room temperature for 1 h. Protein signals were visualized using Pierce\u0026trade; ECL Western Blotting Substrate (Thermo Fisher Scientific, MA, USA) and captured with a ChemiScope 6100 imaging system (Clinx, Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoffuorescence (IF) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence studies, cells were fixed with 4% formaldehyde, permeabilized using 0.5% Triton X-100 (Sigma-Aldrich), and blocked with Immunol Staining Blocking Buffer (Beyotime). Cells were incubated overnight with primary antibodies and subsequently treated with fluorophore-conjugated secondary antibodies (Cell Signaling Technology, MA, USA) for 1 h. Finally, cells were mounted using ProLong Gold Antifade Mountant containing DAPI (Thermo Fisher Scientific) before imaging.\u003c/p\u003e\n\u003cp\u003eFor multiplex immunofluorescence analysis of PDOs and mouse HNSC tissues, sections were stained with antibodies against La/SSB, Ki-67, and cleaved caspase-3. Immunoreactivity was detected using FITC- and Cy3-conjugated secondary antibodies (Cell Signaling Technology, MA, USA), followed by nuclear counterstaining with DAPI (Beyotime). To assess Ki-67 and cleaved caspase-3 expression, a minimum of three sections per HNSCC lesion were evaluated. In each section, more than 150 tumor cells were manually counted, and the numbers of Ki-67\u0026ndash; or cleaved caspase-3\u0026ndash;positive cells were recorded. The proportion of marker-positive cells was determined by dividing the number of positive cells by the total number of tumor cells, and the mean value across all sections was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLentivirus infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll lentiviral constructs were purchased from GenePharma (Shanghai, China), including LV4 plasmids encoding La/SSB, FSCN1, and TFAP2C cDNAs, as well as an empty vector control. In addition, the LV-2 N lentiviral shRNA vector was employed to knock down La/SSB, FSCN1, and TFAP2C, alongside a scrambled shRNA control (shSc). Target sequences are provided in \u003cstrong\u003eSupplementary Table S9\u003c/strong\u003e. Lentiviral production and the establishment of stable cell lines were performed as described previously[19].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHNSCC cells were seeded into 96-well plates at 1.5 \u0026times; 10\u0026sup3; cells per well. Cell viability was assessed at 24, 48, 72, and 96 hours using the Cell Counting Kit-8 (CCK-8; Topscience, Shanghai, China). After a 2-hour incubation with the CCK-8 reagent at 37\u0026deg;C, absorbance was recorded at 450 nm to determine the optical density (OD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdherent cells were harvested and counted prior to seeding. Approximately 1,000 cells were plated per well in 6-cm culture dishes in triplicate and allowed to grow for 14\u0026ndash;16 days. After incubation, the colonies were washed with PBS, fixed in 4% paraformaldehyde, and stained with 0.5% crystal violet (Beyotime, Jiangsu, China). Excess dye was gently washed away with water. Only colonies comprising more than 50 cells were counted and included in subsequent analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEdU staining assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor EdU incorporation assays, 4 \u0026times; 10⁴ cells were plated in each well of 24-well plates and treated with 50 \u0026mu;M EdU for 2 h at 37\u0026deg;C. Following fixation and permeabilization, EdU labeling was performed using the Cell-Light EdU Apollo488 Kit (RiboBio, Guangzhou, China) according to the manufacturer\u0026rsquo;s protocol. Images were acquired using an LSM880+225 Airyscan confocal microscope (Carl Zeiss). The proliferation rate was calculated as the proportion of EdU-positive cells relative to DAPI-stained nuclei.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell migration and invasion assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranswell assays were conducted to evaluate cell migration and invasion. For invasion experiments, the upper surface of the Transwell inserts was coated with 40 \u0026mu;L of Matrigel (Corning, NY, USA). A total of 2 \u0026times; 10⁴ cells in 200 \u0026mu;L of serum-free medium were added to the upper chamber, while the lower chamber was filled with medium containing 20% FBS. After 24 hours of incubation at 37\u0026deg;C, cells on the lower side of the membrane were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell apoptosis analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell apoptosis was evaluated using the Annexin V-APC/PI Apoptosis Detection Kit (Keygen, Jiangsu, China) according to the manufacturer\u0026rsquo;s instructions. In brief, 3 \u0026times; 10⁵ adherent cells were harvested with EDTA-free trypsin and washed twice with PBS. Cells were resuspended in 500 \u0026mu;L Binding Buffer and incubated with 5 \u0026mu;L Annexin V-APC and 5 \u0026mu;L propidium iodide (PI) in the dark for 10 minutes. The percentage of apoptotic cells was determined by flow cytometry (Beckman Coulter, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment and culture of HNSCC organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHNSCC organoids were generated and cultured with minor modifications to previously reported protocols. Fresh HNSCC tumor specimens were washed three times with ice-cold PBS (5 minutes per wash) and cut into 1\u0026ndash;3 mm\u0026sup3; pieces on ice. The tissue fragments were digested with trypsin (Sigma-Aldrich) for about 30 minutes. Once the suspension became turbid, it was passed through a 100-\u0026mu;m cell strainer and centrifuged at 200 \u0026times; g for 5 minutes to collect cell clusters. The collected cell clusters were washed three times with PBS to remove residual enzymes and then embedded in Matrigel (Corning). After the Matrigel had solidified, HNSCC organoid culture medium (BioGenous, Jiangsu, China) was added, and cultures were maintained at 37\u0026deg;C in a CO₂ incubator, with medium refreshed every 3\u0026ndash;5 days.\u003c/p\u003e\n\u003cp\u003eFor lentiviral transduction, HNSCC organoids were first dissociated into small cell clusters using a pipette. The clusters were collected by centrifugation at 200 \u0026times; g for 5 minutes at 4\u0026deg;C and resuspended in culture medium (BioGenous) containing the designated lentiviral particles. The suspension was subjected to spin infection at 700 \u0026times; g for 90 minutes at 25\u0026deg;C, followed by incubation at 37\u0026deg;C for 4 hours. After a brief centrifugation at 300 \u0026times; g for 5 minutes, the cell clusters were re-embedded in Matrigel. Organoid morphology was imaged, and diameters were measured using image analysis software (Photoshop, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA sequencing (RNA-seq) was performed by LC Sciences (Hangzhou, China). Briefly, cDNA was converted to double-stranded DNA, followed by PCR amplification and 2 \u0026times; 150 bp paired-end sequencing (PE150) on an Illumina Novaseq\u0026trade; 6000. The resulting reads were aligned and assembled using StringTie with default parameters to generate FPKM values. Differential gene expression between shLa/SSB FaDu cells and their control counterparts was analyzed using the edgeR package in R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eATAC-seq was carried out with support from Igenebook (Wuhan, China). Nuclei were isolated from 50,000 cells, washed, and resuspended in nuclear lysis buffer. Chromatin accessibility was assessed via transposition using Tn5 transposase, which fragments and tags open chromatin regions. The transposed DNA was purified using the Qiagen MinElute PCR Purification Kit (Qiagen, Hilden, Germany) and amplified by PCR with unique barcodes. PCR products were further cleaned to remove residual primers and adapter dimers, and library quality and concentration were checked using an Agilent Bioanalyzer. Sequencing was performed on an Illumina NovaSeq\u0026trade; platform to obtain 150 bp paired-end reads. Raw sequencing data were processed using a standard bioinformatics pipeline, including alignment, peak calling, and differential chromatin accessibility analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReporter constructs and luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 190-bp fragment of the human FSCN1 gene (-1339/-1139), which includes the TFAP2C-binding site, was amplified from human genomic DNA by PCR. The resulting PCR product was inserted into the pGL4.23[luc2/minP] luciferase reporter vector (Promega) using homologous recombination. The primers used for amplification are listed below: forward, 5\u0026apos;-TAACTGGCCGGTACCGTTCTGGGGCTCAAGGCCCT-3\u0026apos;; reverse, 5\u0026apos;-CTTGATATCCTCGAGGGCCGGGCACTGAGATAACT-3\u0026apos;. A mutant reporter plasmid (pFSCN1^mut-Luc) was generated by altering the TFAP2C-binding site using the Q5 Site-Directed Mutagenesis Kit (NEB, MA, USA) with the following primers: forward, 5\u0026apos;-GGGCTAACACAGGCTCGGACCAGC-3\u0026apos;; reverse, 5\u0026apos;-CATTTAATCCCAGCCAGGGGCTTTC-3\u0026apos;. HEK-293T cells were seeded in 24-well plates and co-transfected with 200 ng of either FSCN1\u003csup\u003ewt\u003c/sup\u003e-Luc or pFSCN1^mut-Luc, 200 ng of pcDNA3.1-TFAP2C or empty pcDNA3.1 vector, and 10 ng of the internal control plasmid pRL-TK. Luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega), and firefly luciferase signals were normalized to Renilla luciferase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin immunoprecipitation (ChIP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe binding of TFAP2C to the La/SSB gene was assessed by ChIP using a TFAP2C-specific antibody. ChIP assays were carried out following the protocol provided with the SimpleChIP\u0026reg; Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, MA, USA). Primers used for PCR and qPCR to amplify the predicted TFAP2C-binding sites within the human La/SSB locus are listed in \u003cstrong\u003eSupplementary Table S10\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-seq data and corresponding clinical information for HNSC patients were obtained from The Cancer Genome Atlas (TCGA, http://cancergenome.nih.gov/). Survival analyses were conducted using Kaplan\u0026ndash;Meier curves and the log-rank test. Patients in the TCGA-HNSC cohort were stratified into high- and low-expression groups according to the median expression levels of TFAP2C, La/SSB, or FSCN1. Gene expression correlations were assessed by Spearman\u0026rsquo;s correlation, and receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value of these genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; standard deviation (SD). For comparisons between two groups, Student\u0026rsquo;s t-test was used, whereas one-way or two-way ANOVA was employed for analyses involving multiple groups. Statistical analyses were performed using GraphPad Prism 9.4.1. P values less than 0.05 were considered statistically significant (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. La/SSB is upregulated in HNSCC and predicts poor patient prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePan-cancer analysis of TCGA data indicated that La/SSB expression was markedly increased in various tumor types, including HNSCC (Fig. S1A). To clarify its clinical significance, \u003cem\u003eLa/SSB\u003c/em\u003e expression was compared between tumor and adjacent nonmalignant (ANM) tissues using TCGA, GSE127165, and GSE178537 datasets. All datasets consistently demonstrated marked upregulation of \u003cem\u003eLa/SSB\u003c/em\u003e in HNSCC tissues (Fig. 1A–C).\u003c/p\u003e\n\u003cp\u003eKaplan–Meier survival analysis demonstrated that patients with higher La/SSB expression had shorter overall survival (OS) and disease-specific survival (DSS) (Fig. 1D–E; Tables 1–2). Western blot analysis of 12 paired HNSCC and ANM samples revealed that La/SSB protein levels were elevated in the majority of tumor tissues (Fig. 1F). IHC staining of an expanded cohort of 60 HNSCC specimens further verified this finding, with significantly higher H-scores observed in tumor tissues compared with adjacent mucosa. Importantly, La/SSB levels were higher in tumors with advanced T stage (T3/T4 compared with T1/T2; Fig. 1G–H) and were associated with reduced overall survival (OS) (Fig. 1I).\u003c/p\u003e\n\u003cp\u003eAt the cellular level, quantitative RT-PCR and western blotting revealed substantially higher La/SSB expression in HNSCC cell lines (FaDu, TU177, TU686, TU212, LIU-LSC-1) compared with NOK (Fig. 1J). Immunofluorescence imaging confirmed predominant nuclear localization of La/SSB in HNSCC cells (Fig. 1K). Taken together, these findings support the role of La/SSB as an oncogenic biomarker that is linked to tumor progression and poor clinical outcomes in HNSCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. La/SSB enhances proliferation, migration, and apoptosis resistance of HNSCC cells in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the functional consequences of La/SSB dysregulation, loss- and gain-of-function experiments were performed. Stable knockdown of La/SSB was achieved in FaDu cells using shRNAs (shLa/SSB#1 and shLa/SSB#2), whereas TU177 cells were engineered to overexpress La/SSB. Western blotting and qRT-PCR analyses confirmed the effective modulation of La/SSB expression (Fig. 2A–B, Fig. S2A–B).\u003c/p\u003e\n\u003cp\u003eCCK-8, colony formation, and EdU assays demonstrated that depletion of La/SSB substantially inhibited the proliferation of HNSCC cells, while its overexpression significantly enhanced growth (Fig. 2C–D, Fig. S2C–E). Given the ability of PDOs to recapitulate tumor heterogeneity, we next evaluated La/SSB function in HNSCC PDO models. Consistent with the cell line data, organoids derived from La/SSB-overexpressing cells displayed larger diameters and stronger Ki-67 staining, whereas La/SSB depletion inhibited growth and reduced proliferative indices (Fig. 2E–I).\u003c/p\u003e\n\u003cp\u003eTranswell assays showed that silencing La/SSB markedly impaired the migratory and invasive abilities of FaDu cells, whereas overexpression enhanced these phenotypes in TU177 cells (Fig. 2J–K). Flow cytometric analysis further showed that La/SSB silencing increased apoptotic cell populations, while its overexpression conferred apoptosis resistance (Fig. S2F–G). Together, these findings establish La/SSB as a potent promoter of HNSCC cell proliferation, migration, and survival in vitro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e \u003cstrong\u003eLa/SSB promotes tumor growth and metastasis in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the tumor-promoting function of La/SSB in vivo, CDX models were generated in nude mice using FaDu cells with stable La/SSB knockdown or TU177 cells overexpressing La/SSB. Tumor growth was monitored over four weeks. Silencing La/SSB significantly delayed tumor growth and reduced tumor volume and weight, whereas La/SSB overexpression markedly accelerated tumor progression (Fig. 3A–D; Fig. S3A–D). IHC analysis revealed lower Ki-67 and higher cleaved caspase-3 levels in La/SSB-deficient tumors, whereas the opposite pattern was observed in La/SSB-overexpressing xenografts (Fig. 3E–H; Fig. S3E–H).\u003c/p\u003e\n\u003cp\u003eFurthermore, tail vein injection assays demonstrated that La/SSB knockdown dramatically decreased the number of pulmonary metastatic nodules, whereas La/SSB overexpression enhanced lung colonization (Fig. 3I–L). These results confirm that La/SSB promotes tumor growth and metastasis of HNSCC in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Integrated transcriptomic and epigenomic profiling identifies FSCN1 as a downstream effector of La/SSB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the molecular mechanisms driving La/SSB-mediated tumorigenesis, RNA sequencing (RNA-seq) and ATAC sequencing (ATAC-seq) were performed in FaDu cells after La/SSB knockdown. RNA-seq analysis revealed 2,813 differentially expressed genes (DEGs) with |log₂FC| \u0026gt; 1 and p \u0026lt; 0.05, including 1,521 downregulated and 1,292 upregulated transcripts (Fig. 4A; Table S1). ATAC-seq revealed substantial changes in chromatin accessibility after La/SSB depletion, with 7,973 peaks differentially enriched (Fig. 4B; Table S2).\u003c/p\u003e\n\u003cp\u003eIntegrative analysis of downregulated genes and regions with reduced chromatin accessibility, combined with upregulated genes from the TCGA-HNSC dataset, yielded seven overlapping candidates (Fig. 4C). Among these, \u003cem\u003eFSCN1\u003c/em\u003e expression was most strongly correlated with poor patient survival (p \u0026lt; 0.01; Fig. 4G, Fig. S4A–F). Consistent with these findings, FSCN1 mRNA expression was markedly elevated in HNSCC tissues compared with ANM across both TCGA and GEO datasets (Fig. 4D–F).\u003c/p\u003e\n\u003cp\u003eATAC-seq tracks visualized using Integrative Genomics Viewer (IGV) indicated markedly reduced chromatin accessibility at the \u003cem\u003eFSCN1\u003c/em\u003e promoter following La/SSB knockdown (Fig. 4H). Correspondingly, In FaDu cells with La/SSB silencing, FSCN1 mRNA and protein levels were decreased, whereas in TU177 cells overexpressing La/SSB, FSCN1 expression was elevated (Fig. 4I–J). IHC staining of clinical samples confirmed co-upregulation of La/SSB and FSCN1, Higher FSCN1 expression was correlated with advanced T stage and reduced overall survival (Fig. 4K–L). Collectively, these results identify FSCN1 as a key downstream effector transcriptionally regulated by La/SSB in HNSCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. FSCN1 mimics and mediates the oncogenic functions of La/SSB in HNSCC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate whether \u003cem\u003eFSCN1\u003c/em\u003e independently exhibits oncogenic activity similar to that of La/SSB, we first generated HNSCC cell lines with stable \u003cem\u003eFSCN1\u003c/em\u003e knockdown or overexpression (Fig. S5A). In FaDu cells, knockdown of FSCN1 significantly suppressed cell proliferation, colony formation, and DNA synthesis, as shown by CCK-8, colony formation, and EdU assays, whereas overexpression of FSCN1 in TU177 cells led to enhanced proliferation and DNA synthesis. (Fig. S5B–E).\u003c/p\u003e\n\u003cp\u003eIn PDO models, \u003cem\u003eFSCN1\u003c/em\u003e depletion significantly reduced organoid size and growth rate, while \u003cem\u003eFSCN1\u003c/em\u003e overexpression enhanced organoid expansion and Ki-67 positivity (Fig. S5F–G). Transwell assays demonstrated that silencing FSCN1 reduced cell migration and invasion, while FSCN1 overexpression enhanced these properties (Fig. S5H–I). Flow-cytometric analysis revealed that FSCN1 silencing increased apoptosis, whereas FSCN1 overexpression conferred apoptosis resistance (Fig. S5J–K). These results indicate that FSCN1 phenocopies the tumor-promoting roles of La/SSB in vitro.\u003c/p\u003e\n\u003cp\u003eIn line with the in vitro results, xenograft studies showed that FSCN1 depletion significantly decreased tumor size and weight, whereas FSCN1 overexpression promoted tumor growth (Fig. S6A–D, S6I–L). Immunohistochemical staining of xenograft tissues confirmed that FSCN1-deficient tumors exhibited decreased Ki-67 and increased cleaved caspase-3 levels, while FSCN1-overexpressing tumors showed the opposite pattern (Fig. S6E–H, S6M–P). Together, these data establish that FSCN1 recapitulates La/SSB-driven oncogenic behavior both in vitro and in vivo.\u003c/p\u003e\n\u003cp\u003eTo further determine whether FSCN1 is essential for La/SSB-induced tumorigenesis, rescue experiments were performed by re-expressing FSCN1 in La/SSB-knockdown FaDu cells (Fig. 5A–B). Restoration of FSCN1 expression significantly rescued the proliferative, migratory, and invasive deficiencies induced by La/SSB loss, as shown by CCK-8, colony-formation, EdU, and Transwell assays (Fig. 5C–E, 5G). The increase in apoptosis caused by La/SSB depletion was also reversed upon FSCN1 re-expression (Fig. 5F).\u003c/p\u003e\n\u003cp\u003eIn vivo, FSCN1 re-expression in La/SSB-deficient FaDu cells restored xenograft tumor growth and lung-metastatic colonization, accompanied by elevated Ki-67 and reduced cleaved caspase-3 expression (Fig. 5H–N). Collectively, these results demonstrate that FSCN1 not only phenocopies but also mediates the tumor-promoting functions of La/SSB, serving as a key downstream effector in HNSCC progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. TFAP2C directly activates FSCN1 transcription downstream of La/SSB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delineate the transcriptional mechanism linking La/SSB to \u003cem\u003eFSCN1\u003c/em\u003e, we performed integrated transcription-factor prediction and correlation analyses. Among candidate regulators identified from JASPAR and ChIP-Atlas databases, in the TCGA-HNSC cohort, TFAP2C exhibited the highest positive correlation with FSCN1 expression (r = 0.214, P \u0026lt; 0.001). (Fig. 6A–C). TFAP2C transcript and protein levels were significantly elevated in HNSCC tissues compared with adjacent mucosa, and higher expression was associated with advanced clinical stage and reduced overall survival (Fig. 6D–E).\u003c/p\u003e\n\u003cp\u003eTo test whether TFAP2C directly regulates \u003cem\u003eFSCN1\u003c/em\u003e, we modulated TFAP2C expression in HNSCC cells. Silencing TFAP2C in FaDu cells markedly decreased \u003cem\u003eFSCN1\u003c/em\u003e mRNA and protein, whereas TFAP2C overexpression in TU177 cells induced robust \u003cem\u003eFSCN1\u003c/em\u003e up-regulation (Fig. 6F). In PDOs, TFAP2C depletion suppressed organoid growth and proliferation, while TFAP2C overexpression enhanced these features, mirroring La/SSB-dependent phenotypes (Fig. 6G–H).\u003c/p\u003e\n\u003cp\u003eBioinformatic motif analysis revealed a high-affinity TFAP2C-binding site within the \u003cem\u003eFSCN1\u003c/em\u003e promoter region (Fig. 6I). Chromatin immunoprecipitation (ChIP) assays confirmed TFAP2C enrichment at this site (Fig. 6J–K). Dual-luciferase reporter assays further demonstrated that TFAP2C significantly increased \u003cem\u003eFSCN1\u003c/em\u003e promoter activity, In contrast, mutation of the binding site eliminated this effect (Fig. 6L). Importantly, La/SSB knockdown diminished TFAP2C occupancy at the \u003cem\u003eFSCN1\u003c/em\u003e promoter, whereas La/SSB overexpression enhanced it (Fig. 6M). Together, these data indicate that La/SSB promotes \u003cem\u003eFSCN1\u003c/em\u003e transcription by facilitating TFAP2C recruitment to its promoter, forming a transcriptional activation axis critical for HNSCC progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Inhibition or loss of La/SSB enhances cisplatin sensitivity in PDOs and La/ssb\u003csup\u003ecKO\u003c/sup\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that La/SSB promotes tumor survival, we next investigated whether its inhibition could potentiate the efficacy of CDDP, a first-line chemotherapy for HNSCC. In patient-derived organoids, La/SSB knockdown significantly sensitized tumors to CDDP treatment, producing a synergistic suppression of viability compared with either intervention alone (Fig. 7A–B). Immunofluorescence staining showed pronounced decreases in Ki-67-positive cells and increases in cleaved-caspase-3-positive cells in the combination group, indicating enhanced apoptosis (Fig. 7C–D).\u003c/p\u003e\n\u003cp\u003eTo evaluate the in vivo therapeutic significance, conditional La/ssb\u003csup\u003ecKO\u003c/sup\u003e mice were produced by crossing La/ssb\u003csup\u003efl/fl\u003c/sup\u003e mice with Krt14-Cre/ERT2 transgenic mice, followed by induction with tamoxifen. (Fig. 7E). After 4-NQO-induced oral carcinogenesis, La/ssb\u003csup\u003ecKO\u003c/sup\u003e mice developed markedly fewer and smaller tongue tumors compared with wild-type controls (Fig. 7F). Combination therapy with CDDP further suppressed tumor burden and invasiveness (Fig. 7G). Histologic analysis revealed extensive necrosis and reduced Ki-67 expression in tumors from La/ssb\u003csup\u003ecKO\u003c/sup\u003e + CDDP mice (Fig. 7H).\u003c/p\u003e\n\u003cp\u003eThese findings demonstrate that La/SSB loss profoundly enhances the antitumor efficacy of cisplatin, suggesting that pharmacologic La/SSB inhibition could be exploited to overcome platinum resistance in HNSCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Coordinated activation of the La/SSB–TFAP2C–FSCN1 axis correlates with malignant progression and poor prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo establish the clinical relevance of this regulatory cascade, we analyzed La/SSB, TFAP2C, and FSCN1 expression in 60 paired HNSCC and adjacent normal samples. IHC analysis revealed a strong positive correlation among the three proteins (Fig. 8A), and tumors exhibiting co-upregulation of all three markers displayed significantly higher T stage and lymph-node metastasis rates. ROC curve analysis demonstrated that these genes exhibited high diagnostic performance for differentiating malignant from normal tissues, with AUCs of 0.872 for La/SSB, 0.868 for TFAP2C, and 0.862 for FSCN1 (Fig. 8B). Consistent trends were observed in the TCGA-HNSC cohort (AUC = 0.844, 0.619, 0.948, respectively; Fig. 8C). Representative IHC staining demonstrated that tumor regions with intense La/SSB expression exhibited concurrent nuclear TFAP2C accumulation and elevated cytoplasmic FSCN1 levels (Fig. 8D). Quantitative assessment showed that patients with elevated levels of all three markers exhibited significantly worse overall survival compared with individuals with low or intermediate expression (Fig. 8E). Collectively, these results define a clinically relevant La/SSB–TFAP2C–FSCN1 axis whose coordinated activation drives HNSCC aggressiveness and predicts adverse patient outcomes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study identifies La/SSB as a pivotal oncogenic driver in HNSCC. Through multi-omics integration and multi-model validation, we reveal that La/SSB reprograms tumor transcriptional activity by enhancing TFAP2C-dependent activation of \u003cem\u003eFSCN1\u003c/em\u003e, thereby coupling RNA-binding dynamics with chromatin-based gene regulation. This work expands the functional paradigm of RNA-binding proteins (RBPs) beyond their canonical post-transcriptional roles, positioning La/SSB as a molecular bridge that links RNA metabolism, cytoskeletal remodeling, and chemotherapeutic resistance in HNSCC.\u003c/p\u003e\n\u003cp\u003eLa/SSB was originally described as an autoimmune antigen in Sjögren syndrome, but accumulating evidence indicates that its dysregulation contributes to tumorigenesis[8, 9, 20-22]. Increased La/SSB expression has been observed in hepatocellular carcinoma, breast cancer, and non-small-cell lung cancer, where it stabilizes oncogenic mRNAs and sustains translation under stress[11, 23-25]. Our findings extend this oncogenic landscape to HNSCC, showing that La/SSB upregulation correlates with advanced disease and poor prognosis. Importantly, its loss attenuates tumor growth and metastatic potential across both in vitro and in vivo systems, underscoring its functional indispensability for HNSCC progression. These data highlight La/SSB as a member of an emerging class of RBPs that exert transcription-like control through selective modulation of chromatin-associated complexes.\u003c/p\u003e\n\u003cp\u003eMechanistically, our integrative RNA-seq and ATAC-seq analyses pinpoint \u003cem\u003eFSCN1\u003c/em\u003e as the dominant downstream effector of La/SSB. \u003cem\u003eFSCN1\u003c/em\u003e encodes an actin-bundling protein essential for the formation of filopodia and invadopodia, cellular structures that enable motility and invasion[26-29]. In normal epithelia, \u003cem\u003eFSCN1\u003c/em\u003e expression is tightly suppressed, whereas its reactivation is a hallmark of metastatic carcinomas[30, 31]. The finding that La/SSB silencing diminishes \u003cem\u003eFSCN1\u003c/em\u003e promoter accessibility, while \u003cem\u003eFSCN1\u003c/em\u003e restoration rescues La/SSB loss-of-function phenotypes, establishes a direct mechanistic dependency. This epistatic relationship reveals a hierarchical control in which La/SSB governs cytoskeletal plasticity through transcriptional remodeling rather than mere mRNA stabilization.\u003c/p\u003e\n\u003cp\u003eUpstream of \u003cem\u003eFSCN1\u003c/em\u003e, we identified TFAP2C as a transcriptional intermediary that translates La/SSB activity into promoter-specific gene activation. TFAP2C is a context-dependent transcription factor known to remodel chromatin accessibility and coordinate oncogenic programs such as epithelial–mesenchymal transition and therapy resistance[32-35]. Prior studies have shown that TFAP2C cooperates with lineage-specific factors like p63 or GATA3 to shape enhancer landscapes in epithelial tumors[36-38]. Our data now suggest that La/SSB may facilitate TFAP2C recruitment to target promoters, potentially by modulating chromatin architecture or RNA–protein scaffold formation. This crosstalk exemplifies a broader principle whereby RBPs indirectly dictate transcriptional outcomes by influencing transcription factor loading and nucleosome accessibility—a conceptual convergence of post-transcriptional and epigenetic regulation.\u003c/p\u003e\n\u003cp\u003eFunctionally, La/SSB also confers protection against genotoxic stress, contributing to CDDP resistance. Platinum compounds induce DNA crosslinks and activate DNA-damage response pathways, leading to apoptosis in sensitive cells. The survival advantage conferred by La/SSB likely stems from its ability to sustain translation of DNA-repair and anti-apoptotic transcripts during stress adaptation. Inhibition or deletion of La/SSB markedly enhanced CDDP efficacy in organoid and conditional-knockout models, suggesting that targeting La/SSB may disrupt this adaptive translational network. These findings align with prior reports that RBPs such as IGF2BP2 or YBX1 mediate similar resistance programs, reinforcing the idea that translational plasticity constitutes a major determinant of chemoresistance in epithelial malignancies[39].\u003c/p\u003e\n\u003cp\u003eConceptually, the La/SSB–TFAP2C–FSCN1 axis delineated here underscores a multilayer regulatory circuit in which an RNA-binding protein indirectly orchestrates transcriptional activation to promote cytoskeletal reorganization, invasion, and therapeutic evasion. This dual control over RNA fate and chromatin accessibility may represent a general strategy exploited by tumors to sustain malignant phenotypes under environmental and therapeutic stress. From a translational perspective, La/SSB’s tumor-restricted expression and multifunctional role render it an attractive candidate for therapeutic intervention. Pharmacologic or RNA-based La/SSB inhibition, particularly when combined with platinum-based chemotherapy, could enhance treatment responses in aggressive HNSCC. Furthermore, La/SSB and \u003cem\u003eFSCN1\u003c/em\u003e expression signatures may serve as biomarkers for risk stratification and therapeutic guidance.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study uncovers a noncanonical regulatory axis centered on La/SSB that integrates RNA-binding and chromatin-modulating functions to drive HNSCC progression and chemoresistance. These findings broaden our understanding of how RBPs rewire oncogenic signaling and open new avenues for therapeutic targeting of RBP-dependent vulnerabilities in epithelial cancers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eHNSC\u003c/strong\u003e: Head and Neck Squamous Cell Carcinoma\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANM\u003c/strong\u003e:\u0026nbsp;adjacent non-malignant tissues\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLa/SSB\u003c/strong\u003e: Small RNA binding exonuclease protection factor La\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFSCN1\u003c/strong\u003e: Fascin actin-bundling protein 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTFAP2C\u003c/strong\u003e: Transcription factor activating protein 2 gamma\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDDP\u003c/strong\u003e: Cisplatin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDMSO\u003c/strong\u003e: Dimethyl Sulfoxide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBS\u003c/strong\u003e: phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHEK 293T\u003c/strong\u003e: human embryonic kidney 293T\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR\u003c/strong\u003e: quantitative real-time PCR\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCK-8\u003c/strong\u003e: Cell Counting Kit-8\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePDO\u003c/strong\u003e: patient-derived Oragnoid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDX\u003c/strong\u003e: cell line-derived xenograft model\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNOK\u003c/strong\u003e: Normal oral epithelial cell line\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATCC\u003c/strong\u003e: American Type Culture Collection\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDEGs\u003c/strong\u003e: Differentially expressed genes\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDARs\u003c/strong\u003e: Differentially accessible regions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4NQO\u003c/strong\u003e: 4-nitroquinoline N-oxide\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATAC\u003c/strong\u003e: Assay for Transposase Accessible Chromatin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGV\u003c/strong\u003e: Integrative Genomics Viewer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIF\u003c/strong\u003e: immunofluorescence\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChIP\u003c/strong\u003e: chromatin immunoprecipitation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026amp;E\u003c/strong\u003e: Hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIHC\u003c/strong\u003e: immunohistochemistry\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esiRNAs\u003c/strong\u003e: small interfering RNA\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eshRNA\u003c/strong\u003e: short hairpin RNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-seq\u003c/strong\u003e: RNA sequencing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTCGA\u003c/strong\u003e: The Cancer Genome Atlas\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCoIP\u003c/strong\u003e: Co-immunoprecipitation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOS\u003c/strong\u003e: Overall survival\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTFs\u003c/strong\u003e: Transcription factors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROC\u003c/strong\u003e: Receiver Operating Characteristic\u003cbr\u003e\u003cstrong\u003eAUC\u003c/strong\u003e: Area Under the Curve\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study can be accessed from the GEO repository under accession numbers GSE283322 and GSE283323.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of China (82171127, 82371133, 82171128 and 82303021), Anhui Provincial Natural Science Foundation (2208085MH239), the Natural Science Foundation of Universities of Anhui Province (2022AH051134), Discipline Construction Project of the First Affiliated Hospital of Anhui Medical University (NO. 4245) and Anhui Medical University Foundation（2023xkj146).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSYM XJZ and YFH supervised the project; SXL, SYM, XJZ and YFH designed the experiments; SXL, WTZ and WWL performed the experiments; SXL, WTZ, DSC, JP, HL, JJZ, XMW, MJZ, MJW, CQC, SCL, XLY, QX and GYH analyzed the data; SXL, XJZ and DSC wrote the paper; SYM and YFH revised the paper. All authors read and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial registration:\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Clinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollection of clinical samples for IHC, western blot, and qRT-PCR analyses was approved by the Research Ethics Committee of the First Affiliated Hospital of Anhui Medical University (PJ 2021-02-32), and PDO experiments were approved under PJ 2023-12-38. All animal studies, except for PDX models, complied with the university’s Experimental Animal Ethical Committee guidelines (LLSC20211380). All participants provided written informed consent prior to sample collection.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e 2023; 73: 17\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGessain G, Anzali A-A, Lerousseau M, Mulder K, Bied M, Auperin A \u003cem\u003eet al\u003c/em\u003e. TREM2-Expressing Multinucleated Giant Macrophages Are a Biomarker of Good Prognosis in Head and Neck Squamous Cell Carcinoma. \u003cem\u003eCancer Discov\u003c/em\u003e 2024; 14: 2352\u0026ndash;2366.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao H, Lan T, Kuang S, Wang L, Li J, Li Q \u003cem\u003eet al\u003c/em\u003e. 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TKI resistant-based prognostic immune related gene signature in LUAD, in which FSCN1 contributes to tumor progression. \u003cem\u003eCancer Lett\u003c/em\u003e 2022; 532: 215583.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie F, Huang C, Liu F, Zhang H, Xiao X, Sun J \u003cem\u003eet al\u003c/em\u003e. CircPTPRA blocks the recognition of RNA N6-methyladenosine through interacting with IGF2BP1 to suppress bladder cancer progression. \u003cem\u003eMol Cancer\u003c/em\u003e 2021; 20: 68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Chen S, Jiang Q, Deng J, Cheng F, Lin Y \u003cem\u003eet al\u003c/em\u003e. TFAP2C facilitates somatic cell reprogramming by inhibiting c-Myc-dependent apoptosis and promoting mesenchymal-to-epithelial transition. \u003cem\u003eCell Death Dis\u003c/em\u003e 2020; 11: 482.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMughal MJ, Zhang Y, Li Z, Zhou S, Peng C, Zhang Y-Q \u003cem\u003eet al\u003c/em\u003e. TFAP2C-DDR1 axis regulates resistance to CDK4/6 inhibitor in breast cancer. \u003cem\u003eCancer Lett\u003c/em\u003e 2024; 610: 217356.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang M, Zhong A, Liu H, Zhao L, Wang Y, Lu Z \u003cem\u003eet al\u003c/em\u003e. EZH2 loss promotes gastric squamous cell carcinoma. \u003cem\u003eNat Commun\u003c/em\u003e 2025; 16: 6032.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuirico L, Rizzolio S, Bertone S, Cirillo PDR, Savino A, Vitale N \u003cem\u003eet al\u003c/em\u003e. The chimeric aptamer axl-miR-214sponge inhibits breast cancer and melanoma dissemination. \u003cem\u003eMol Ther\u003c/em\u003e 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGantner CW, Weatherbee BAT, Wang Y, Zernicka-Goetz M. Assembly of a stem cell-derived human postimplantation embryo model. \u003cem\u003eNat Protoc\u003c/em\u003e 2024; 20: 67\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Jia W, Luo Z, Li Y, Liu H, Fu L \u003cem\u003eet al\u003c/em\u003e. VGLL1 cooperates with TEAD4 to control human trophectoderm lineage specification. \u003cem\u003eNat Commun\u003c/em\u003e 2024; 15: 583.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePattison JM, Melo SP, Piekos SN, Torkelson JL, Bashkirova E, Mumbach MR \u003cem\u003eet al\u003c/em\u003e. Retinoic acid and BMP4 cooperate with p63 to alter chromatin dynamics during surface epithelial commitment. \u003cem\u003eNat Genet\u003c/em\u003e 2018; 50: 1658\u0026ndash;1665.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShuai Y, Ma Z, Yue J, Li C, Ju J, Wang X \u003cem\u003eet al\u003c/em\u003e. MNX1-AS1 suppresses chemosensitivity by activating the PI3K/AKT pathway in breast cancer. \u003cem\u003eInt J Biol Sci\u003c/em\u003e 2025; 21: 3689\u0026ndash;3704.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Univariate and multivariate Cox regression analyses of the correlation between clinical characteristics with overall survival\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"614\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnivariate analysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMultivariate analysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal(N)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHazard ratio (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHazard ratio (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026lt;= 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026gt; 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.088 (0.770 - 1.538)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0.632\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e1.134 (0.794 - 1.619)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.489\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e355\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.968 (0.652 - 1.439)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0.874\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e0.942 (0.628 - 1.415)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.775\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHistologic grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e463\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eG1\u0026amp;G2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e347\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eG3\u0026amp;G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.055 (0.715 - 1.557)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0.788\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e0.986 (0.666 - 1.460)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.944\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eClinical stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e464\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eStage I\u0026amp;Stage II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eStage III\u0026amp;Stage IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e357\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.161 (0.759 - 1.775)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0.492\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e1.091 (0.705 - 1.687)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.695\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eSSB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e239\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e239\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.812 (1.270 - 2.587)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e1.924 (1.337 - 2.771)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe bold values are defined as significant (p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Univariate and multivariate Cox regression analyses of the correlation among the clinical characteristics with disease-specific survival\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"614\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnivariate analysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMultivariate analysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal(N)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHazard ratio (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHazard ratio (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e503\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026lt;= 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026gt; 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.262 (0.964 - 1.653)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0.090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e1.349 (1.015 - 1.791)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e503\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e369\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.760 (0.571 - 1.012)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0.061\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e0.772 (0.571 - 1.043)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.092\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eHistologic grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e483\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eG1\u0026amp;G2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e362\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eG3\u0026amp;G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.942 (0.690 - 1.286)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0.706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e0.894 (0.653 - 1.225)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.487\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eClinical stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e489\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eStage I\u0026amp;Stage II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eStage III\u0026amp;Stage IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.226 (0.885 - 1.700)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0.221\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e1.191 (0.849 - 1.671)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.312\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eSSB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e503\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003eReference\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 72px;\"\u003e\n \u003cp\u003e252\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.563 (1.193 - 2.049)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e1.717 (1.296 - 2.276)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe bold values are defined as significant (p \u0026lt; 0.05).\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"HNSCC, La/SSB, TFAP2C, FSCN1, tumor progression, cisplatin resistance","lastPublishedDoi":"10.21203/rs.3.rs-8395850/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8395850/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eHead and neck squamous cell carcinoma (HNSCC) is marked by aggressive behavior, a paucity of effective treatment strategies, and unsatisfactory survival rates. The RNA-binding protein La/SSB is frequently overexpressed in diverse cancers, but its biological function and mechanistic contribution to HNSCC pathogenesis remain unclear.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eBy combining transcriptome-wide RNA sequencing and chromatin accessibility analyses with functional experiments conducted both in vitro and in vivo, we sought to define the role of La/SSB in HNSCC. Patient-derived organoids (PDOs), cell-derived xenografts (CDXs), and conditional La/SSB knockout (La/ssb\u003csup\u003ecKO\u003c/sup\u003e) mice were used to validate its tumorigenic and therapeutic relevance.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eLa/SSB expression was substantially elevated in HNSCC tissues and cell lines, with higher levels being associated with reduced overall survival. Functionally, La/SSB promoted tumor cell proliferation, invasion, and resistance to cisplatin. Integrative multi-omics analysis identified \u003cem\u003eFSCN1\u003c/em\u003e as a key downstream effector transcriptionally activated by TFAP2C. Mechanistically, La/SSB enhanced TFAP2C recruitment and chromatin accessibility at the \u003cem\u003eFSCN1\u003c/em\u003e promoter, thereby establishing a La/SSB\u0026ndash;TFAP2C\u0026ndash;FSCN1 regulatory axis that sustains oncogenic transcriptional programs. Inhibition or genetic deletion of La/SSB significantly sensitized tumors to cisplatin both in PDOs and \u003cem\u003eLa/ssb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eThe La/SSB\u0026ndash;TFAP2C\u0026ndash;FSCN1 axis promotes HNSCC progression and chemoresistance by coupling RNA-binding activity with transcriptional reprogramming. Therapeutic modulation of La/SSB may offer a promising approach to improve cisplatin responsiveness in the management of HNSCC.\u003c/p\u003e","manuscriptTitle":"The RNA-binding protein La/SSB drives head and neck squamous cell carcinoma progression through TFAP2C-mediated transcriptional activation of FSCN1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 04:21:32","doi":"10.21203/rs.3.rs-8395850/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-02-11T15:31:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-09T13:44:08+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-02T14:27:12+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-26T20:53:24+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-22T14:19:39+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-15T17:33:26+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-15T17:20:47+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-15T17:10:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-04T14:57:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-18T13:14:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2025-12-18T13:14:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d91f5965-2011-445f-827d-9816ed97382f","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61204445,"name":"Biological sciences/Cancer/Head and neck cancer"},{"id":61204446,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2026-05-06T16:22:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 04:21:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8395850","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8395850","identity":"rs-8395850","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}