RESULTS: ARG2 promotes tumorigenesis by increasing cellular proliferation, migration, invasion and vasculogenic mimicry in GBM cells, at least in part due to overexpression of MMP2/9. The nor-NOHA significantly reduced migration and tube formation of ARG2-overexpressing cells. HCMV immediate-early proteins (IE1/2) or its downstream pathways upregulated the expression of ARG2 in U-251 MG cells. Immunostaining of GBM tissue sections confirmed the overexpression of ARG2, consistent with data from subsets of Gene Expression Omnibus. Moreover, higher levels of ARG2 expression tended to be associated with poorer survival in GBM patient by analyzing data from TCGA.

METHODS: The role of ARG2 in tumorigenesis was examined by proliferation-, migration-, invasion-, wound healing- and tube formation assays using an ARG2-overexpressing cell line and ARG inhibitor, N (omega)-hydroxy-nor-L-arginine (nor-NOHA) and siRNA against ARG2 coupled with functional assays measuring MMP2/9 activity, VEGF levels and nitric oxide synthase activity. Association between HCMV and ARG2 were examined in vitro with 3 different GBM cell lines, and ex vivo with immunostaining on GBM tissue sections. The viral mechanism mediating ARG2 induction was examined by siRNA approach. Correlation between ARG2 expression and patient survival was extrapolated from bioinformatics analysis on data from The Cancer Genome Atlas (TCGA).

MATERIALS AND METHOD: Eighty-seven paraffin-embedded ameloblastoma cases (20 unicystic, 47 solid/multicystic, 3 desmoplastic and 17 recurrent) were subjected to immunohistochemistry for expression of cortactin, N-WASP, WIP, Src kinase and F-actin, and findings correlated with clinicopathological parameters.

RESULTS: Invadopodia proteins (except Src kinase) and F-actin were widely detected in ameloblastoma (cortactin: n = 73/87, 83.9%; N-WASP: n = 59/87; 67.8%; WIP: n = 77/87; 88.5%; and F-actin: n = 87/87, 100%). Protein localization was mainly cytoplasmic and/or membranous, and occasionally nuclear for F-actin. Cortactin, which functions as an actin-scaffolding protein, demonstrated significantly higher expression levels within ameloblastoma tumoral epithelium than in stroma (P < 0.05). N-WASP, which coordinates actin polymerization and invadopodia-mediated extracellular matrix degradation, was overexpressed in the solid/multicystic subtype (P < 0.05). WIP, an upstream regulator of N-WASP, and F-actin were significantly upregulated along the tumour invasive front compared to tumour centres (P < 0.05). Except for males with cortactin overexpression, other clinical parameters (age, ethnicity and anatomical site) showed no significant correlations.

METHODS: Gene expression of IL-6 and IL-6Rα in PSC and PDAC cells was measured with qRT-PCR. The role of PSC-secreted IL-6, JAK/Stat3 signaling, and Nrf2 mediation on EMT-related genes expression was also examined with qRT-PCR. EMT phenotypes were assessed with morphological change, wound healing, migration, and invasion.

RESULTS: PSC expressed higher mRNA levels of IL-6 but lower IL-6Rα compared to PDAC cells. Neutralizing IL-6 in PSC secretion reduced mesenchymal-like morphology, migration and invasion capacity, and mesenchymal-like gene expression of N-cadherin, vimentin, fibronectin, collagen I, Sip1, Snail, Slug, and Twist2. Inhibition of JAK/Stat3 signaling induced by IL-6 repressed EMT and Nrf2 gene expression. Induction of Nrf2 activity by tert-butylhydroquinone (tBHQ) increased both EMT phenotypes and gene expression (N-cadherin, fibronectin, Twist2, Snail, and Slug) repressed by IL-6 neutralizing antibody. Simultaneous inhibition of Nrf2 expression with siRNA and Stat3 signaling further repressed EMT gene expression, indicating that Stat3/Nrf2 pathway mediates EMT induced by IL-6.

CONCLUSIONS: IL-6 from PSC promotes EMT in PDAC cells via Stat3/Nrf2 pathway.