Dataset Information

Cdh1 Proteomic Analysis - A Synthetic Lethal Interaction between APC/C and Topoisomerase Poisons Uncovered by Proteomic Screens

ABSTRACT: The Anaphase-promoting complex/cyclosome (APC/C) cofactor Cdh1 modulates proliferation and cell cycle exit by targeting multiple cell cycle regulators for ubiquitin-dependent degradation. Lack of Cdh1 results in structural and numerical chromosome aberrations, a hallmark of genomic instability, although the specific targets involved in these defects are not well understood. Here, we use MS-based proteomics to identify substrates of Cdh1 important for cell cycle regulation. To this end, we performed three experiments: 1 - SILAC analysis of Cdh1 deficient asynchronous MEFs (samples were fractionated by means of SDS-PAGE and IEF) 2 - iTRAQ analysis of Cdh1 deficient synchronous MEFs (samples were fractionated by means of IEF) 3 - iTRAQ analysis of brain tissue in a Cdh1 deficient Mouse (samples were fractionated by means of IEF) LC-MS was carried out using a LTQ-Orbitrap Velos as described in Eguren et al. iTRAQ raw data was processed using Proteome Discoverer 1.3 interfaced with Mascot 2.2 SILAC raw data was processed using MaxQuant suite using Andromeda as search engine Further details can be found in Eguren et al.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Fernando Garcia

PROVIDER: PXD000511 | Pride | 2014-02-20

REPOSITORIES: Pride

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Publications

A synthetic lethal interaction between APC/C and topoisomerase poisons uncovered by proteomic screens.

Eguren Manuel M Álvarez-Fernández Mónica M García Fernando F López-Contreras Andrés J AJ Fujimitsu Kazuyuki K Yaguchi Hiroko H Luque-García José Luis JL Fernández-Capetillo Oscar O Muñoz Javier J Yamano Hiroyuki H Malumbres Marcos M

Cell reports 20140206 4

The Anaphase-promoting complex/cyclosome (APC/C) cofactor Cdh1 modulates cell proliferation by targeting multiple cell-cycle regulators for ubiquitin-dependent degradation. Lack of Cdh1 results in structural and numerical chromosome aberrations, a hallmark of genomic instability. By using a proteomic approach in Cdh1-null cells and mouse tissues, we have identified kinesin Eg5 and topoisomerase 2α as Cdh1 targets involved in the maintenance of genomic stability. These proteins are ubiquitinated ...[more]

PMID: 24508461

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Project description:Background: E-cadherin is an adherens junction protein that forms homophilic intercellular contacts in epithelial cells while also interacting with the intracellular cytoskeletal networks. It has roles including establishment and maintenance of cell polarity, differentiation, migration and signalling in cell proliferation pathways. Its downregulation is commonly observed in epithelial tumours and is a hallmark of the epithelial to mesenchymal transition (EMT). Methods: To improve our understanding of how E-cadherin loss contributes to tumorigenicity, we investigated the impact of its elimination from the non-tumorigenic breast cell line MCF10A. We performed cell-based assays and whole genome RNAseq to characterize an isogenic MCF10A cell line that is devoid of CDH1 expression due to an engineered homozygous 4bp deletion in CDH1 exon 11. Results: The E-cadherin-deficient line, MCF10A CDH1-/- showed subtle morphological changes, weaker cell-substrate adhesion, delayed migration, but retained cell-cell contact, contact growth inhibition and anchorage-dependent growth. Within the cytoskeleton, the apical microtubule network in the CDH1-deficient cells lacked the radial pattern of organization present in the MCF10A cells and F-actin formed thicker, more numerous stress fibres in the basal part of the cell. Whole genome RNAseq identified compensatory changes in the genes involved in cell-cell adhesion while genes involved in cell-substrate adhesion, notably ITGA1, COL8A1, COL4A2 and COL12A1, were significantly downregulated. Key EMT markers including CDH2, FN1, VIM and VTN were not upregulated although increased expression of proteolytic matrix metalloprotease and kallikrein genes was observed. Conclusions: Overall, our results demonstrated that E-cadherin loss alone was insufficient to induce an EMT or enhance transforming potential in the non-tumorigenic MCF10A cells but was associated with broad transcriptional changes associated with tissue remodelling. Examination of the impact of E-cadherin (CDH1) loss in an isogenic pair of breast cell lines.

Dataset Information

Cdh1 Proteomic Analysis - A Synthetic Lethal Interaction between APC/C and Topoisomerase Poisons Uncovered by Proteomic Screens

Publications

A synthetic lethal interaction between APC/C and topoisomerase poisons uncovered by proteomic screens.

Similar Datasets

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