Differential roles of kallikrein-related peptidase 6 in malignant transformation and DeltaNp63beta-mediated epithelial-mesenchymal transition of oral squamous cell carcinoma.

We previously reported that epithelial-to-mesenchymal transition (EMT) was mediated by DeltaNp63beta in oral squamous cell carcinoma (OSCC). In this study, DNA microarray analyses were performed using DeltaNp63beta-overexpressing OSCC cells to identify genes associated with DeltaNp63beta-mediated EMT. Thereby, we focused on kallikrein-related peptidase (KLK) 6, most up-regulated following DeltaNp63beta-overexpression, that activates protease-activated receptors (PARs). In RT-PCR analyses, DeltaNp63 was positively associated with KLK6 and PAR2 and negatively with PAR1 in OSCC cells. By DeltaNp63 knockdown, KLK6 and PAR2 expression was decreased and PAR1 was increased. Furthermore, KLK6 knockdown led to enhancing migration and invasion, and inhibiting proliferation, suggesting EMT-phenotypes. Although, in the KLK6 or PAR2 knockdown cells, phosphorylation of ERK was reduced, it was restored in the KLK6 knockdown OSCC cells treated with recombinant KLK6 proteins. Immunohistochemistry showed DeltaNp63, KLK6, and PAR2 were more strongly expressed in the epithelial dysplasia and central region of OSCC than normal oral epithelium, whereas PAR1 expression was undetectable. Interestingly, at the invasive front of OSCC, DeltaNp63, KLK6, and PAR2 were reduced, but PAR1 was elevated. In addition, the OSCC patients with decreasing KLK6 expression at the invasive front had more unfavourable prognosis. These results suggested differential roles of KLK6 in malignant transformation and EMT; high DeltaNp63beta expression up-regulates KLK6-PAR2 and down-regulates PAR1, inducing malignant transformation in oral epithelium with stimulating proliferation through ERK signal activation. Moreover, KLK6-PAR2 expression is down-regulated and PAR1 is up-regulated when DeltaNp63beta expression is decreased, leading to EMT with enhancing migration and invasion through ERK signal reduction at the invasive front.