Epithelial-to-mesenchymal transition (EMT) in intraductal papillary mucinous neoplasm (IPMN) is associated with high tumor grade and adverse outcomes.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is generally associated with increased tumor aggressiveness and poor prognosis. We evaluated EMT characteristics in intraductal papillary mucinous neoplasm (IPMN) tumor specimens and their potential role as biomarkers for malignancy, metastasis, and adverse patient outcomes. METHODS: IPMN surgical specimens were identified and reviewed by two gastrointestinal pathologists. Immunohistochemical analysis of E-cadherin, vimentin, and ZEB-1 was performed. Samples were linked to clinicopathologic and outcome data for these patients. Western blot test was used to evaluate ZEB-1 expression in IPMN samples; 846 human miRNAs were profiled, and EMT-related differentially expressed miRNAs were validated using quantitative real-time polymerase chain reaction. RESULTS: Fifty-eight IPMN specimens and five normal pancreatic tissue samples were immunohistochemically stained and scored. E-cadherin expression was significantly lower in malignant versus low-grade IPMN (p < 0.05). Vimentin expression was increased in malignant IPMN tumor samples (p < 0.05). EMT was associated with increased lymph node metastasis and decreased survival of malignant IPMN patients (p < 0.05). ZEB-1, an imperative EMT regulator, was exclusively expressed by malignant IPMN tumors. miRNA hierarchical clustering demonstrated grouping of two main IPMN subgroups: low-grade IPMN versus high-grade IPMN and carcinoma. Twenty-four miRNAs were differentially expressed (14 up-regulated, 10 down-regulated). The EMT-regulatory miRNAs, miR-200c and miR-141, were down-regulated (twofold and 1.8-fold decrease, respectively) in malignant versus low-grade IPMN (p < 0.05). CONCLUSIONS: EMT may play a role in IPMN tumorigenesis and metastasis. EMT molecular deregulations could be utilized as potential novel biomarkers for the identification of high-risk IPMN patients.

Use of fluorescent substrates for characterization of Gaucher disease mutations.

Gaucher disease results from impaired activity of the lysosomal enzyme beta-glucocerebrosidase. More than 200 mutations within the glucocerebrosidase gene have been associated with this disease. In this study we tested the effect of several mutations (K157Q, D140H, E326K, D140H+E326K, V394L and R463C) on RNA stability, protein stability and activity toward four different fluorescent substrates (LR-12-GC, Bodipy-12-GC, LR-0-PAP-glucose and 4-MUG), using the vaccinia-derived expression system. The results indicated that the K157Q mutation leads to RNA instability, causing low protein levels and a concomitant reduction in beta-glucocerebrosidase activity. All other tested mutations led to production of glucocerebrosidase RNA and protein with stabilities comparable to those of the normal counterpart. The D140H variant exhibited a high activity toward the tested substrates while the variant enzymes containing either the E326K or D140H and E326k mutations together expressed low beta-glucocerebrosidase activity. The V394L variant exhibited low activity toward the tested substrates, while a higher activity was presented by the R463C containing glucocerebrosidase variant. Our results strongly indicated that the LR-12-GC substrate distinguishes between severities of different mutant glucocerebrosidase variants overexpressed in a heterologous system.