RNA sequencing analysis of ESRP1, 2 overepxressing PANC-1 and MIA-PaCa2
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PROVIDER: PRJNA1152095 | ENA |
REPOSITORIES: ENA
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Project description:Epithelial splicing regulatory proteins (ESRPs) are splicing regulators known to play a critical role in epithelial-mesenchymal transition (EMT) and the progression of various cancers, including pancreatic ductal adenocarcinoma (PDAC). Numerous cancer-related genes, such as CD44, FGFR2, and CDNND1, are spliced by ESRP1 and 2 with differential expression of variants. Here, we analyzed ESRP1 and 2 overexpressing pancreatic cancer cells, PANC-1 and MIA-PaCa2, and discovered splicing targets of ESRPs.
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Project description:Inhibition of the dual function cell cycle and transcription kinase CDK7 is known to affect the viability of cancer cells, but the mechanisms underlying cell line-specific growth control remain poorly understood. Here, we employed a previously developed, highly specific small molecule inhibitor that non-covalently blocks ATP binding to CDK7 (LDC4297) to study the mechanisms underlying cell line-specific growth using a panel of genetically heterogeneous human pancre-atic tumor lines as model system. Although LDC4297 diminished both transcription rates and CDK T-loop phosphorylation in a comparable manner, some PDAC lines displayed significantly higher sensitivity than others. We focused our analyses on two well-responsive lines (Mia-Paca2 and Panc89) that, however, showed significant differences in their viability response to extended exposure to limiting LDC4297 concentrations. Biochemical and RNAseq analysis revealed strik-ing differences in gene expression and cell cycle control. Especially the downregulation of a group of cell cycle control genes, among them CDK1/2 and CDC25A/C, correlated well to the observed viability differences in Panc89 versus Mia-Paca2 cells. A parallel downregulation of regulatory pathways supported the hypothesis of a feedforward programmatic effect of CDK7 inhibitors, eventually causing hypersensitivity of PDAC lines.
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Project description:A summary of the work associated to these microarrays is the following: The need for an integrated view of all data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize phenomena in terms of how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed Biological Association Networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). Seven cell lines representative of different types of cancer including colon cancer (HT29 and Caco2), breast cancer (MCF7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by microarrays covering the whole human genome and analyzed with the GeneSpring GX software package, v.7.3.1. Genes deregulated in common in the two colon cancer cell lines studied, were subject of Biological Association Networks construction. Dikkopf homolog-1 (DKK1) was a clear node of this network, and functional validations of this target using a siRNA showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of differentially expressed genes in the two breast cancer cell lines studied. siRNA treatment against UGT1A showed also an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was a gene overexpressed in common among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. Biological Association Networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using iRNA technology against these three genes show chemosensitization toward MTX. Two cell lines are compared, which are MIA PaCa2 pancreatic cancer cells sensitive to methotrexate and MIA PaCa2 cells resistant to 10e-6M MTX. Six samples are provided which correspond to triplicates of each cell line. The samples provided were analyzed using the specific software GeneSpring GX.
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