Sympathetic innervation contributes to perineural invasion of salivary adenoid cystic carcinoma via the β2-adrenergic receptor

Purpose: Perineural invasion (PNI) is reported to correlate with local recurrence and poor prognosis of salivary adenoid cystic carcinoma (SACC). However, the pathogenesis of PNI remains unclear. The aims of this study were to investigate the correlation between sympathetic innervation and SACC PNI and to elucidate how the sympathetic neurotransmitter norepinephrine (NE) regulates the PNI process. Materials and methods: Sympathetic innervation and beta 2-adrenergic receptor (beta 2-AR) expression in SACC tissues were evaluated by immunohistochemistry. The NE concentrations in SACC tissues and dorsal root ganglia (DRG) coculture models were measured by ELISA. beta 2-AR expression in SACC cells was detected by performing quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence assay. SACC cells were treated with NE, the nonselective alpha-AR blocker phentolamine, the beta 2-AR antagonist ICI118,551, or were transfected with beta 2-AR small interfering RNA (siRNA). Proliferation was evaluated in methyl thiazolyl tetrazolium assay, and migration was evaluated in Transwell assay and wound-healing assay. PNI was tested through both Transwell assay and a DRG coculture model. The expressions of epithelial-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) were measured by performing qRT-PCR and Western blot assay. Results: Sympathetic innervation and beta 2-AR were highly distributed in SACC tissues and correlated positively with PNI (P=0.035 and P=0.003, respectively). The sympathetic neurotransmitter NE was overexpressed in SACC tissues and DRG coculture models. Exogenously added NE promoted proliferation, migration, and PNI of SACC cells via beta 2-AR activation. NE/beta 2-AR signaling may promote proliferation, migration, and PNI by inducing EMT and upregulating MMPs. However, beta 2-AR inhibition with either an antagonist or siRNA abrogated NE-induced PNI. Conclusion: Collectively, our findings reveal the supportive role of sympathetic innervation in the pathogenesis of SACC PNI and suggest beta 2-AR as a potential therapeutic target for treating PNI in SACC.