Pancreatic ductal adenocarcinoma (PDAC) is a devastating metastatic disease for which better therapies are urgently needed. Macrophages enhance metastasis in many cancer types, however, the role of macrophages in PDAC liver metastasis remains poorly understood. Here we found that PDAC liver metastasis critically depends on the early recruitment of granulin secreting inflammatory monocytes to the liver. Mechanistically, we demonstrate that granulin secretion by metastasis associated macrophages (MAMs) activates resident hepatic stellate cells (hStCs) into myofibroblasts that secrete periostin, resulting in a fibrotic microenvironment that sustains metastatic tumour growth. Disruption of MAM recruitment or genetic depletion of granulin reduced hStCs activation and liver metastasis. Interestingly, we found that circulating monocytes and hepatic MAMs in PDAC patients express high levels of granulin. These findings suggest that recruitment of granulin expressing inflammatory monocytes plays a key role in PDAC metastasis and may serve as a potential therapeutic target for PDAC liver metastasis.

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Oligosaccharide moieties of glycoproteins are structurally altered during development, carcinogenesis, and malignant transformations. It is well known that ␤1-6 GlcNAc branching, a product of UDP-GlcNAc ␣-mannoside ␤1-6-N-acetylglucosaminyltransferase (GnT-V), is associated with malignant transformation as the results of such alterations. However, the mechanism by which ␤1-6 GlcNAc branching is linked to metastasis remains unclear, because the identification of specific glycoprotein(s) that are glycosylated by GnT-V and its biological function have not been examined. We herein report that matriptase, which activates both urokinase-type plasminogen activator and hepatocyte growth factor, is a target protein for GnT-V. The overexpression of GnT-V in gastric cancer cells leads to severe peritoneal dissemination in athymic mice, which can be attributed to the increased expression of matriptase. This increase was due to the acquired resistance of matriptase to degradation, since it is glycosylated by GnT-V and a corresponding increase in the active form. These results indicate that this process is a key element in malignant transformation, as the direct result of oligosaccharide modification.N-Glycans are widely distributed on cell surfaces and secreted glycoproteins, where structural change is observed in development, carcinogenesis, and malignant transformation (1-3). Recent findings suggest that the structural changes in N-glycans are one of the critical steps for cellular transformation and are directly linked to malignant transformation. Previous studies have revealed that ␤1-6 GlcNAc branching on N-glycans, a product of UDP-GlcNAc ␣-mannoside ␤1-6-Nacetylglucosaminyltransferase (GnT-V 1 ; EC 2.4.1.155), is a key structure associated with tumor metastasis and malignant transformation (4 -6). Since we reported on the purification and cDNA cloning of human 8), numerous studies have reported that ␤1-6 GlcNAc branching is associated with malignant transformation, including tumor invasion and metastasis (9 -12). Gene transcription of GnT-V is regulated by proto-oncogenes such as the Ets family (13, 14), src (15) and erbB2 (16). The sequence analysis of the 5Ј-flanking region of GnT-V revealed the functional binding sites of the Ets family. In addition, certain transcription factors belonging to the Ets family are activated by the Ras-Raf-mitogen-activated protein kinase signaling pathway, which leads to cell proliferation and transformation (17). The Ras proto-oncogene sustains activating mutations in ϳ20% of all human tumors. Ras signaling is induced by other common mutations, such as the amplification of Neu/ErbB-2 in breast cancer (18). These findings suggest that elevated GnT-V activity in human tumors might commonly occur at the level of gene expression.A recent study using GnT-V knockout mice demonstrated that the expression of GnT-V is essential for tumor growth and metastasis (12). The author reported that GnT-V stimulated membrane ruffling and phosphatidylinositol 3-kinase-protein kinase B activation. Howeve...

Human glycoproteins exhibit enormous heterogeneity at each N-glycosite, but few studies have attempted to globally characterize the site-specific structural features. We have developed Integrated GlycoProteome Analyzer (I-GPA) including mapping system for complex N-glycoproteomes, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. Using an N-glycopeptide database that we constructed, we created novel scoring algorithms with decoy glycopeptides, where 95 N-glycopeptides from standard α1-acid glycoprotein were identified with 0% false positives, giving the same results as manual validation. Additionally automated label-free quantitation method was first developed that utilizes the combined intensity of top three isotope peaks at three highest MS spectral points. The efficiency of I-GPA was demonstrated by automatically identifying 619 site-specific N-glycopeptides with FDR ≤ 1%, and simultaneously quantifying 598 N-glycopeptides, from human plasma samples that are known to contain highly glycosylated proteins. Thus, I-GPA platform could make a major breakthrough in high-throughput mapping of complex N-glycoproteomes, which can be applied to biomarker discovery and ongoing global human proteome project.