The Key Vitamin A Regulator RBP1 Modulates Malignant Biological Behaviors in Head and Neck Squamous Cell Carcinoma

Background Head and neck squamous cell carcinoma (HNSCC) ranks as the sixth most common malignancy globally. The involvement of vitamins in carcinogenesis has attracted growing research interest, with vitamin A (retinol) being extensively implicated in cancer biology. Mounting evidence suggests that dysregulated expression of retinol-binding proteins profoundly impacts tumor initiation and progression by compromising retinoid signaling integrity within the tumor microenvironment (TME). Cellular retinol-binding protein 1 (RBP1, also known as CRBP1), a critical modulator of intracellular retinol metabolism in the retinoid family, is primarily responsible for retinol trafficking and metabolic processing. Although RBP1 has been characterized in multiple cancer types and is closely associated with vitamin A homeostasis, its mechanistic role in HNSCC pathogenesis and its regulatory effects on malignant cellular phenotypes remain elusive. Objectives This study aimed to systematically investigate the pathogenic mechanisms of HNSCC and to evaluate the potential of RBP1 as a novel molecular target driving HNSCC development and progression. These findings may provide a foundation for developing effective early diagnostic biomarkers and targeted therapeutic strategies for HNSCC. Methods Publicly available head and neck squamous cell carcinoma (HNSCC) transcriptomic and clinical datasets were retrieved from The Cancer Genome Atlas (TCGA) database. Differentially expressed protein-coding genes associated with poor prognosis were identified through bioinformatics screening. In parallel, RNA sequencing (RNA-seq) was performed on three paired tissue specimens (tumor and adjacent non-tumor) from HNSCC patients. Genes involved in vitamin A (retinol) metabolism and signaling were curated from the GeneCards database. The intersection of these four gene sets—TCGA-derived prognostic genes, RNA-seq differentially expressed genes, vitamin A-associated genes, and protein-coding genes—was computed to identify candidate molecules. Cellular retinol-binding protein 1 (RBP1, also known as CRBP1) emerged as the most significantly correlated gene with HNSCC and was associated with unfavorable prognosis.The mRNA and protein expression levels of RBP1 were quantified across HNSCC cell lines by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis, respectively. Based on endogenous RBP1 expression profiles, appropriate HNSCC cell lines were selected for subsequent functional investigations.A retrospective cohort comprising 63 HNSCC patients was enrolled, with paired tumor and adjacent normal tissue specimens and comprehensive clinical metadata collected. RBP1 protein expression was assessed by immunohistochemistry (IHC). Univariate and multivariate Cox regression analyses were conducted on both TCGA cohort data and the 63-patient clinical dataset to evaluate the association between RBP1 expression and malignant phenotypes as well as overall survival outcomes.Gain- and loss-of-function models were established through RBP1 overexpression using the pcDNA3.1 vector and short interfering RNA (siRNA)-mediated knockdown in selected HNSCC cell lines. Functional assays were performed as follows: Cell Counting Kit-8 (CCK-8) assay and colony formation assay for proliferative capacity; wound healing assay for migratory potential; and Transwell chamber assay (with or without Matrigel coating) for invasive capability.Drug resistance analysis was performed using R software and the Gene Expression Profiling Interactive Analysis (GEPIA) platform to investigate the correlation between RBP1 expression and chemoresistance in HNSCC. Tumor immune infiltration patterns were analyzed via the Tumor Immune Estimation Resource (TIMER) database to evaluate RBP1-associated immune microenvironment landscapes in HNSCC. Results Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis demonstrated that cellular retinol-binding protein 1 (RBP1, also known as CRBP1) expression was significantly elevated in head and neck squamous cell carcinoma (HNSCC) cell lines compared with normal head and neck epithelial cell lines. Immunohistochemistry (IHC) revealed markedly higher RBP1 protein levels in HNSCC tumor tissues than in adjacent normal tissues. Clinical correlation analysis of The Cancer Genome Atlas (TCGA) data indicated that regional lymph node metastasis (N stage) and age were significantly associated with RBP1 expression ( P < 0.05). Univariate Cox regression analysis identified primary tumor size and local extent (T stage), regional lymph node metastasis (N stage), and high RBP1 expression as predictors of poor prognosis in HNSCC patients ( P < 0.05). Variables with P < 0.05 in the univariate analysis were subsequently entered into multivariate Cox regression analysis, which confirmed that high RBP1 expression served as an independent prognostic factor for unfavorable outcomes in HNSCC. Analysis of the 63-patient clinical cohort revealed significant correlations between RBP1 expression and regional lymph node metastasis (N stage) as well as overall TNM stage ( P < 0.05). Univariate Cox analysis demonstrated that primary tumor size and local extent (T stage), regional lymph node metastasis (N stage), advanced TNM stage, and high RBP1 expression were associated with poor prognosis ( P < 0.05). Multivariate Cox analysis of these significant variables identified advanced TNM stage and high RBP1 expression as independent prognostic factors for adverse outcomes in HNSCC patients. In RBP1-overexpressing (pcDNA3.1-RBP1) SCC-9 cells, proliferative capacity, colony formation efficiency, migratory ability, and invasive potential were significantly enhanced ( P < 0.001). Conversely, in RBP1-knockdown (siRNA-RBP1) SCC-25 cells, these malignant phenotypes were markedly attenuated ( P < 0.001). Bioinformatics analysis using R software (version 4.4.2) and the Gene Expression Profiling Interactive Analysis (GEPIA) platform revealed a significant correlation between elevated RBP1 expression and paclitaxel resistance in HNSCC, which was further associated with unfavorable prognosis. Tumor immune infiltration analysis via the Tumor Immune Estimation Resource (TIMER 2.0) database demonstrated that RBP1 expression was significantly associated with the infiltration levels of B cells, CD4+ T cells, CD8+ T cells, dendritic cells, M2 macrophages, mast cells, monocytes, tumor-associated macrophages (TAMs), T follicular helper (Tfh) cells, T helper 1 (Th1) cells, and T helper 2 (Th2) cells, as well as immune checkpoint molecules. Notably, RBP1 exhibited a negative regulatory role in these immune interactions ( P < 0.05). Conclusion Cellular retinol-binding protein 1 (RBP1, also known as CRBP1), a critical regulator of vitamin A (retinol) metabolism, is significantly upregulated in head and neck squamous cell carcinoma (HNSCC) tissues and cell lines. Elevated RBP1 expression is clinically correlated with aggressive malignant phenotypes, including advanced primary tumor size and local extent (T stage), regional lymph node metastasis (N stage), higher overall TNM stage, and patient age, and serves as an independent predictor of unfavorable prognosis. Functional assays demonstrate that RBP1 overexpression (pcDNA3.1-RBP1) promotes proliferative capacity, migratory ability, and invasive potential in HNSCC cells, whereas RBP1 knockdown (siRNA-RBP1) markedly suppresses these malignant behaviors. Furthermore, bioinformatics analysis reveals that RBP1 is significantly associated with paclitaxel resistance and poor clinical outcomes. Notably, RBP1 participates in immune regulation within the HNSCC tumor microenvironment (TME), exerting a negative modulatory effect on antitumor immune responses. Collectively, these findings establish RBP1 as a valuable biomarker for lymph node metastasis and a potential therapeutic target influencing HNSCC initiation, progression, and prognosis. RBP1 holds considerable promise as a novel early diagnostic biomarker and druggable target for precision management of HNSCC.