Radiotherapy-associated Furin Expression and Tumor Invasiveness in Recurrent Laryngeal Cancer.

Abstract

Recurrent laryngeal cancer often shows an aggressive phenotype after radiotherapy and does not respond to conventional therapeutic strategies. In this study, we investigated the contribution of furin to cellular invasiveness in radio-resistant laryngeal cancer. Using previously established AMC-HN-3 and AMC-HN-8 cell lines from laryngeal carcinoma patients, recurrent laryngeal cancer models were generated by cumulative irradiation (AMC-HN-3-70Gy and AMC-HN-8-70Gy). Immunocytochemistry and western blotting were used to determine the epithelial-mesenchymal transition (EMT). Invasion capacity was assessed using an in vitro invasion assay. Zymography was used to assess metalloproteinase-2 (MMP-2) activity. Tumor xenografts were developed to compare growth rate and furin expression in vivo. Furin expression in 35 patients (45 samples) with salvage total laryngectomy after radiation-based treatment was assessed by laryngeal cancer tissue microarray. Both AMC-HN-3-70Gy and AMC-HN-8-70Gy cell lines underwent EMT following radiation. However, AMC-HN-3-70Gy cells showed increased cellular invasiveness, whereas AMC-HN-8-70Gy cells showed no difference. AMC-HN-3-70Gy cells also exhibited elevated furin expression with up-regulated expression of the active form of membrane type 1-matrix metalloproteinase (MT1-MMP)/MMP-2, whereas AMC-HN-8-70Gy cells did not show significant changes. After administration of a furin inhibitor (chloromethyl ketone (CMK)), AMC-HN-3-70Gy cells showed a significant decrease in MT1-MMP/MMP-2 expression and cellular invasiveness. Nine of 22 samples (40.9%) from salvage total laryngectomy and one of 13 pre-radiation samples (7.7%) had high furin expression. Post-radiation, furin expression increased in seven of 10 patients whose pre- and post-radiation samples were available; all-cancer mortality (three patients) was observed in this group. Together with EMT, furin activity may serve as an indicator of an aggressive cancer phenotype, suggesting that furin is a potentially useful target for recurrent laryngeal cancer.