ABSTRACT: We used time-dependent spheroidal cultures of pancreatic cancer cells to investigate how spatiotemporal regulation in spheroids induces epithelial-mesenchymal plasticity (EMP). We generated a round bottom spheroidal model using β3 integrin-negative pancreatic cancer cells in 2D conventional monolayer cultures, and compared the expression of integrin signaling-related genes that induce spheroidal geometry to determine if spheroidal geometry induces integrin switching. To identify the potential genes whose expression changed in response to culture conditions, we performed microarray analysis of gene expression in human MIA PaCa-2 cells grown under monolayer and spheroid culture conditions for 6 days and 13 days.
Project description:We used time-dependent spheroidal cultures of pancreatic cancer cells to investigate how spatiotemporal regulation in spheroids induces epithelial-mesenchymal plasticity (EMP). We generated a round bottom spheroidal model using β3 integrin-negative pancreatic cancer cells in 2D conventional monolayer cultures, and compared the expression of integrin signaling-related genes that induce spheroidal geometry to determine if spheroidal geometry induces integrin switching. Overall design: To identify the potential genes whose expression changed in response to culture conditions, we performed microarray analysis of gene expression in human MIA PaCa-2 cells grown under monolayer and spheroid culture conditions for 6 days and 13 days.
Project description:Integrin β3 is seen as a key anti-angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro- or anti-angiogenic depends on the context in which it is expressed. To understand precisely β3’s role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3-dependent adhesome. We show that depletion of β3-integrin in this cell type leads to changes in microtubule behaviour that control their migration. β3-integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2 driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3-integrin levels are reduced.
Project description:miR-145 is downregulated in multiple cancers. The introduction of miR-145 could alleviate the tumor burden in the pancreatic cancer mouse model. However, how miR-145 mediates the tumor suppression is still an open question. In this study, we aimed to identify the targets of miR-145 using a SILAC approach.
Project description:A microarray approach was used to measure and compare whole genome expression in non-sorted cell population and in subsets of cultured cells, including stem-like cancer cell and non-stem like cancer cell populations, MiaPaCa-2 pancreatic cancer cells from culture were sorted by Fluorescence Activated Cell Sorting (FACS) using 3 markers: CD44, CD133 and EpCAM. Three resulting populations, i.e., not sorted, sorted triple positive and sorted triple negative, were analyzed. 4 cultures were sorted independently to arrive at 4 biological replicates.
Project description:Pancreatic ductal adenocarcinoma (PDAC) has one of the worst prognoses of any human malignancy and there are few human cellular models of disease progression. When human PDAC cells are injected into immunodeficient animals, they create tumors of the late stage from which they were derived. We hypothesized that if human pancreatic cancer cells were converted to pluripotency and then allowed to differentiate back into pancreas, the developmental progression would recapitulate early stages of the cancer. To that end, we have generated isogenic matched sets of induced pluripotent stem (iPS) cell-like lines from epithelial cells of human pancreatic tumors and from histologically normal epithelial cells at the resected pancreatic margins. Notably, when injected into immunodeficient mice, at low or high passages, a human pancreatic cancer iPS-like line, but not the corresponding margin iPS-like line, slowly generates intra-epithelial neoplasia (PanIN) ductal structures that typically reflect the early stages of human pancreatic cancer. The PanIN-like ducts can be isolated and cultured. They secrete protein products reflective of PanINs and provide new insights into underlying regulatory networks. An additional iPS-like line from histologically normal cells at a pancreatic resection margin, but containing a mutation that predisposes to PDAC, does not generate PanIN ductal structures. These studies demonstrate that iPS technology can be exploited to recapitulate early progression events of a human epithelial cancer. Study includes a single experiment (#10): a tumor-adjacent pancreatic tissue control (10N); tumor tissue (10C); IPS-transformed tissue control (10N12); and IPS-transformed tumor tissue (10C22).
Project description:KRAS mutations occur in approximately 25% of non-small cell lung cancer (NSCLC). They account for the therapy resistance to EGFR inhibitors and are suggested to be difficult to target by specific drugs. Therefore, new therapies for KRAS mutant NSCLC are urgently needed. The histone H3K4 and H3K9 di/mono-demethylase KDM1A is a key epigenetic writer, aberrantly upregulated in many cancer types, including NSCLC. In order to understand the functional role of KDM1A in the progression of lung adenocarcinoma, KDM1A expression profiles were analysed in tissue microarrays (TMAs) including 182 lung adenocarcinoma. KDM1A expression correlated with high grade and metastasized tumor. To investigate the impact of KDM1A in lung adenocarcinoma development, we used the KRAS mutated A549 cell line to establish a shRNA-mediated stable KDM1A knockdown cell clone. Unexpectedly, KDM1A knockdown had only a slight effect on retardation of cell growth. However, cell invasion and self-renewal capability was significantly decreased by KDM1A inhibition. KDM1A knockdown in A549 cell resulted in a dramatic change in the transcriptome profile as determined by RNA-Seq. Interestingly, genes involved in the KRAS signature and lung epithelial marker genes were significantly affected upon KDM1A knockdown. Ingenuity pathway analysis also suggested that the alternative integrin β3-KRAS signaling axis, which is involved in stem cell like properties, is abrogated upon KDM1A knockdown. Indeed, Integrin β3 and its non-canonical ligand galectin-3 were strongly downregulated and their downstream NF-κB activity was decreased upon KDM1A knockdown. Finally, correlation of KDM1A to the Integrin β3 level was validated in TMAs. Overall design: Determining the role of KDM1A in A549 cells, mRNA profiles of control and knockdown samples of A549 cells, generated by deep sequencing, in triplicate, using Illumina HiSeq 2500.
Project description:Cellular senescence is an important in vivo mechanism that prevents the propagation of damaged cells. However, the precise mechanism(s) regulating senescence are not well characterized. Here, we find that ITGB3 (integrin beta 3 or β3) is epigenetically regulated by the Polycomb protein CBX7. β3 expression accelerates the onset of senescence in human primary fibroblasts, by activating the TGFβ pathway in a cell autonomous and non-cell autonomous manner. β3 levels are dynamically increased during oncogeneinduced senescence (OIS) through CBX7 epigenetic regulation. DNA-damage and therapy-induced senescence (TIS) also induce β3 expression. In fact, downregulation of β3 levels override OIS and TIS, independently of its ligand-binding activity. Moreover, cilengitide, a αvβ3 antagonist, has the ability to block the SASP without affecting proliferation. Finally, we show an increase in β3 levels during aging in mice and humans. Altogether, our data show that integrin β3 subunit is a marker and regulator of senescence.
Project description:Purpose: The goals of this study are to elucidate the influence of integrin β3 signaling on STAT1-dependnet gene expression in IFNγ-treated HSCs. Methods: Wild type (WT) HSCs were cultured with or without IFNγ and/or VN in the presence of stem cell factor (SCF) plus thrombopoietin (TPO). Subsequently, cultured HSC fraction (CD48- c-kit+ Sca-1+ Lineage-) were sorted, followed by mRNA sequence using Ion Proton (n>4). Moreover, to extract genes whose expression were changed via STAT1 in the presence of IFNγ, mRNA profiles of STAT1-/- HSCs treated with or without IFNγ were also generated by the same way. The sequence reads that passed quality filters were analyzed by CLC genomic workbench. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm10) with CLC genomic workbench. Indeed, hierarchical clustering analysis showed that IFNγ-treated STAT1-/- HSCs was categorized to the group including Wt HSCs cultured in the absence of IFNγ rather than HSCs treated with IFNγ. Furthermore, gene set enrichment analysis (GSEA) showed that STAT1-dependent upregulated gene sets were significantly enriched within genes whose expression was enhanced in HSCs treated with VN and IFNγ. In contrast, integrin β3 signaling in the absence of IFNγ appears to not influence the expression of IFNγ/STAT1-dependent genes, as evidenced by the observation that VN treatment was statistically and significantly independent of the enrichment of gene sets that were both up-regulated by STAT1 Conclusions: Our study represents that STAT1 plays a central role in IFNγ-mediated HSC responses and integrin β3 signaling in HSCs promotes STAT1-dependent gene expression in the presence of IFNγ. Overall design: After HSCs derived from wild type (WT) and STAT1-/- mice were treated with IFNg and/or vitronectin for 5 days, mRNA profiles were generated by deep sequencing using Ion Proton system (n>4).
Project description:The aim of our study was to assess the impact of simvastatin on the amount of cytosolic lipid droplets (LDs), which are implicated in many biological processes including proliferation, inflammation, carcinogenesis, apoptosis, necrosis or growth arrest. Human pancreatic cancer cells MiaPaCa-2 were treated with simvastatin (6 and 12 _M) for 24 hours. Changes in expression of genes related to lipid metabolism in the simvastatin-treated cells were examined by DNA microarray analysis. The treatment of the cells with simvastatin increased their intracellular content of LDs, partially due to the uptake of cholesterol and triacylglycerides from medium; but in particular, due to enhanced synthesis of triacylglycerides as proved by detection of significant overexpression of genes related to de novo synthesis of triacylglycerides and phospholipids. Further, simvastatin markedly influenced expression of genes directly affecting cell proliferation and signaling.
Project description:Purpose: Core 3 derived glycans, a major type of O-glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy, because of loss of expression of functional β3-N-acetylglucosaminyltransferase-6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re-expressing core 3 synthase in pancreatic cancer cells that had lost expression. Experimental Design: We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. We therefore re-expressed in human pancreatic cancer cells (Capan-2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Results: Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared with vector control cells. Expression of core 3 O-glycans induced altered expression of β1 integrin, decreased activation of focal adhesion kinase, led to the down regulation of expression of several genes including REG1α and FGFR3, and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. Conclusions: These findings indicate that expression of core 3 derived O-glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins. Two-condition experiment, Core 3 synthase stable expression (C3) vs. vector control (PLVX) cells. Biological replicates: 3 Core 3 synthase stable expression, 3 vector control, independently grown and harvested. One replicate per array.