Project description:GNAS, a gene encoding G-protein stimulating alpha subunit, is frequently mutated in intraductal papillary mucinous neoplasms (IPMNs), which is an indolent and slow-growing pancreatic neoplasm that secretes abundant mucin. GNAS mutation is not observed in conventional ductal adenocarcinomas of the pancreas. To determine the functional significance of GNAS mutation in pancreatic ductal cells, we examined in vitro phenotypes and gene expression profiles of cells of pancreatic ductal lineage, HPDE, PK-8, PCI-35, and MIA PaCa-2, with exogenous expression of either wild-type or mutated (R201H) GNAS. We found that exogenous GNAS upregulated intracellular cyclic-adenine monophosphate, particularly in the mutated GNAS transfectants. Exogenous GNAS induced no obvious cell-growth promotion, but induced suppression in some cells. The exogenous GNAS upregulated MUC2 and MUC5AC in HPDE and PK-8, and the latter was most sensitive to exogenous GNAS, exhibiting drastic alteration of the global gene expression that is consistent with that of IPMN. Hence, PK-8 expressing exogenous mutated GNAS may be an ideal in vitro model of IPMN. On the other hand, exogenous GNAS downregulated expression of mucin genes and produced modest alteration of gene expression profiles in PCI-35 and MIA PaCa-2, indicating lower sensitivity to exogenous GNAS. Furthermore, we showed diverse and cell-type specific mucin expression pathways with complicated interactions between signaling pathways of the G-protein coupled receptor (GPCR), the mitogen-activated protein kinase (MAPK), and the phosphatidylinositol 3 kinase (PI3K), in which the GPCR pathway appeared to be dominant in some and the MAPK pathway in others. In conclusion, mutated GNAS found in IPMNs may extensively alter gene expression profiles, including expression of mucin genes, with the interaction with MAPK and PI3K pathways in pancreatic ductal-lineage cells, which may determine the characteristic phenotype of the neoplasm. Cells of pancreatic cancer cell lines, PK-8, PCI-35,and MIA PaCa-2, were seeded at 4 M-CM-^W 10^5 cells/well in 6-well plates and incubated for 24 hours at 37M-BM-0C in 5% CO2 with humid atmosphere. Then the cells were transfected with either pcDNA 3.1/V5-His vector or pcDNA3.1-GNAS(R201H)-V5-His vector using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturerM-bM-^@M-^Ys recommendations. The cells were incubated for 24 hours and collected by dissociation using trypsin. Total RNAs were isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany). Serial analysis of gene expression (SAGE) library was constructed using a SOLiD SAGE Kit (Life Technologies) according to the manufactureM-bM-^@M-^Ys instruction. The constructed libraries were analysed by means of the massively parallel sequencing method using SOLiD 4 System (Life Technologies). The SAGE analysis was performed for samples obtained from single transfection experiment.

Project description:Intraductal Papillary Mucinous Neoplasms (IPMNs) of the pancreas are cyst-like precursor lesions that give rise to 25% of pancreatic ductal adenocarcinomas (PDAC). While ~90% of cases are diagnosed before cancer forms, metrics and markers by which to determine if an IPMN will progress are currently lacking. Overall, 96% of IPMNs harbor KRAS (~80%) and/or GNAS (~66%) driver mutations and IPMN may be classified as either gastric, intestinal, or pancreatobiliary type. Recently, we identified a shared program of pyloric type metaplasia between pancreatic and gastric injury. Here, we combined immunostaining, RNA-sequencing, molecular biology, and analysis of patient samples to identify major drivers of this program in IPMN. Methods Single cell RNA-sequencing datasets of human IPMN were assayed for markers of pyloric-type metaplasia. 37 IPMN patient samples were immunostained using MxIHC for MUC5AC, CD44v9, and AQP5. KrasG12D and GnasR201C expression was modified in IPMN cell lines to identify transcriptomic programs driven by each oncogene, and in combination. Gene sets were compared to murine and human gastric cell types. Transcriptomic drivers were identified and manipulated in vitro. Candidate driver expression was evaluated by immunostaining of patient IPMN and graded. Results Analysis of published bulk and scRNA-seq IPMN datasets revealed expression of previously described markers of pyloric type metaplasia. Marker expression was confirmed in patient samples. Manipulation of KrasG12D and GnasR201C expression in IPMN cell lines identified the relative contributions of each oncogene to this gastric phenotype. Regulon analysis suggests that transcription factors SPDEF, CREB3L1, and CREB3L4 amplify this program in response to GnasR201C expression. All transcription factors were expressed in patient IPMN samples. Conclusions In response to oncogenic mutation(s), IPMN form in the pancreas and express markers of pyloric type metaplasia. KrasG12D and GnasR201C expression drive this program through independent and synergistic mechanisms that result in amplified expression of mucins and gastrokines, consistent with the phenotype identified in vivo.

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IPMN model mice with Kras Gnas mutation

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