Expressions of NLRP3, Caspase-1, and GSDMD in nasopharyngeal carcinoma tissue and association with recurrence and metastasis

Abstract

Objective: To investigate the expressions of Nod-like receptor protein 3 (NLRP3), cysteine-aspartic acid protease 1 (Caspase-1), and Gasdermin D (GSDMD) in nasopharyngeal carcinoma (NPC), and their relationships with the recurrence and metastasis of NPC. Methods: A retrospective study was conducted on 421 patients diagnosed with NPC between December 2014 and January 2020. The expressions of NLRP3, Caspase-1, and GSDMD in pathological specimens were examined with immunohistochemistry and multiplex immunofluorescence staining. Univariate and multivariate Cox regression analyses were applied to identify the factors influencing NPC recurrence and metastasis. In vitro experiments with NPC cell line HNE-2 were used to explore the functional mechanisms of NLRP3, Caspase-1, and GSDMD. Results: Multivariate Cox analysis revealed that tumor staging of Ⅲ-Ⅳ(HRrecurrence=2.74, 95%CIrecurrence: 1.61-4.65; HRmetastasis=1.90, 95%CImetastasis: 1.04-3.49) and pre-treatment plasma EBV-DNA levels≥1 500 copies/ml (HRrecurrence=1.91, 95%CIrecurrence: 1.13-3.23; HRmetastasis=2.07, 95%CImetastasis: 1.23-3.50)were independent risk factors for NPC recurrence and metastasis, while positive expression of NLRP3(HRrecurrence=0.17, 95%CIrecurrence: 0.08-0.35; HRmetastasis=0.30, 95%CImetastasis: 0.15-0.59), Caspase-1(HRrecurrence=0.32, 95%CIrecurrence: 0.18-0.59; HRmetastasis=0.43, 95%CImetastasis: 0.25-0.76), and GSDMD(HRrecurrence=0.48, 95%CIrecurrence: 0.25-0.91; HRmetastasis=0.96, 95%CImetastasis: 0.53-1.74) served as independent protective factors. Age (HR=1.02, 95%CI: 1.01-1.04) and intensity-modulated radiotherapy (HR=0.51, 95%CI: 0.30-0.88) were independent factors for NPC recurrence, whereas chemotherapy (HR=0.50, 95%CI: 0.29-0.88) acted as an independent protective factor for NPC metastasis (all P<0.05). NPC patients with positive expressions of the three proteins had higher locoregional recurrence-free survival, distant metastasis-free survival, and overall survival compared to those with negative expressions (all P<0.05). In vitro experiments revealed that the overexpression of NLRP3 activated the NLRP3/Caspase-1/GSDMD signaling pathway, as evidenced by Western Blot analysis. Enzyme-linked immunosorbent assay and scanning electron microscopy demonstrated that overexpression of NLRP3 promoted pyroptosis in HNE-2 cells. Cellular functional assays further confirmed that overexpression of NLRP3 significantly inhibited the proliferation, invasion, and migration of HNE-2 cells. Conclusion: Positive expressions of NLRP3, Caspase-1, and GSDMD serves as independent protective factors for recurrence and metastasis of NPC, potentially by promoting cell pyroptosis and thus inhibiting NPC cell proliferation, invasion, and migration.