RAMP1 Promotes Lymphatic Metastasis in Head and Neck Squamous Cell Carcinoma via VEGF-C/PROX-1 Mediated Lymphangiogenesis

Background: Lymph node metastasis (LM) is associated with poor prognosis in head and neck squamous cell carcinoma (HNSCC); however, the molecular drivers of tumor-induced lymphangiogenesis remain incompletely understood. Methods: This study aimed to analyze clinical HNSCC specimens using transcriptomic sequencing to identify differentially expressed genes associated with lymphatic metastasis. Receptor Activity-Modifying Protein 1 (RAMP1) expression was quantified by reverse transcription-quantitative polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blotting. Moreover, functional assays (Transwell, wound healing, Cell Counting Kit-8 (CCK-8), and tube formation) were performed using HNSCC cell lines and human lymphatic endothelial cells. Furthermore, a footpad xenograft mouse model was established to investigate the effects of RAMP1 on tumor growth, lymphangiogenesis, and LM in vivo. Results: RAMP1 was significantly overexpressed in HNSCC tissues with LM. Elevated RAMP1 expression was associated with poor prognosis, increased intratumoral lymphatic vessel density (p < 0.05), and strong predictive power for LM (area under the curve (AUC) = 0.808). In vitro, RAMP1 knockdown reduced proliferation, migration, and invasion of HNSCC cells, and decreased expression of the lymphangiogenic factor vascular endothelial growth factor C (VEGF-C) and its transcriptional regulator Prospero homeobox protein 1 (PROX-1). Conversely, RAMP1 overexpression had opposite effects. RAMP1 also promoted tube formation in lymphatic endothelial cells through a paracrine mechanism. In vivo, RAMP1 overexpression increased lymphangiogenesis and LM in xenograft models, whereas its suppression showed inhibitory effects (p < 0.05). Conclusions: This study indicated that RAMP1 promotes lymphangiogenesis and lymphatic metastasis in HNSCC by regulating the VEGF-C/PROX-1 signaling axis, thereby identifying RAMP1 as a potential pro-metastatic factor and therapeutic target for HNSCC.