Dataset Information

Mapping the spatial proteome of metastatic cells in colorectal cancer

ABSTRACT: Here, we aimed to study metastasis mechanisms using spatial proteomics applied to the KM12 cell model of metastasis. KM12 cells were metabolically labelled using SILAC. SILAC has been successfully used for the analysis of proteome turnover and for the identification of changes in proteome localization as a consequence of DNA damage.10,23-24 Subcellular fractionation of the KM12 cells into five subcellular fractions corresponding to cytoplasm (CEB), plasma, mitochondria and ER/golgi membranes (MEB), nuclear (NEB), chromatin-bound (NEB-CBP) and cytoskeletal proteins (PEB) contributed to clarify the molecular mechanisms underlying CRC metastasis. Protein abundance and localization were measured in parallel for the five separate subcellular fractions, providing a map draft of the spatially deregulated protein complexes and networks in CRC metastasis.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Colon Cancer

SUBMITTER: Alberto Pelaez García

LAB HEAD: Ignacio Casal

PROVIDER: PXD006656 | Pride | 2017-09-11

REPOSITORIES: Pride

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Publications

Mapping the Spatial Proteome of Metastatic Cells in Colorectal Cancer.

Mendes Marta M Peláez-García Alberto A López-Lucendo María M Bartolomé Rubén A RA Calviño Eva E Barderas Rodrigo R Casal J Ignacio JI

Proteomics 20171001 19

Colorectal cancer (CRC) is the second deadliest cancer worldwide. Here, we aimed to study metastasis mechanisms using spatial proteomics in the KM12 cell model. Cells were SILAC-labeled and fractionated into five subcellular fractions corresponding to: cytoplasm, plasma, mitochondria and ER/golgi membranes, nuclear, chromatin-bound and cytoskeletal proteins and analyzed with high resolution mass spectrometry. We provide localization data of 4863 quantified proteins in the different subcellular f ...[more]

PMID: 28861940

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Project description:Snail1 is a transcriptional repressor required for a correct embryonic development. In cancer, Snail1 promotes the epithelial to mesenchymal transition in tumorigenic epithelial cells. In this work, we have analyzed the control of Snail1 in the differentiation of the 3T3-L1 cell line derived from murine embryo cells. The activation by snail of 3T3-L1 induced typical markers of cancer-activated fibroblasts as S100A4 or CD44. We generated 3T3-L1 cells stably over expressing Snail1 (3T3L1/Snail1) and control (3T3-L1/control) cells. We used SILAC quantitative approach to identify and characterize protein alterations induced by Snail1. Cells were fractionated in 5 subcellular fractions. The nuclear fraction of the cells was separated by 10% SDS-PAGE. Gels with forward and reverse experiments were stained with Coomassie Blue and cut into 18 slices prior to reduction, alkylation and digestion with trypsin. Tryptic peptides were scanned and fragmented with a linear ion trap-Orbitrap Velos (ThermoScientific). We identified a total of 3108 proteins, with 2572 quantified proteins, and 565 proteins modulated >1.5-fold by Snail1 overexpression. Among them, we found interesting up-regulated proteins associated to early differentiation of adipogenesis (C/EBPβ) and down-regulated proteins implicated in the final stages of differentiation to adipocytes (Fatty acid-binding protein or Fatty acid synthase). We also observed as down-regulated proteins important mediators of PPARγ pathway. We also observed downregulation of proteins implicated in mTOR, SRC and JAK/STAT pathway. We validated these proteomics data by western blot and qPCR in 3T3-L1 cells and other types of fibroblasts with capable to differentiate to terminal mesenchymal phenotypes, as well as in mesenchymal stem cells (MSC). This work provided insight into novel proteins with potential roles in the regulation of differentiation of the 3T3-L1 and MSCs as Nr2F6, ASC-1, Prrx1 or Cbx6. These candidates are down regulated due to the overexpression of Snail1 in 3T3-L1 cells. We next investigated the potential binding of Snail1 to promoter of these candidates. In silico analysis with MatInspector program revealed various putative E-box consensus motifs for Snail1. We performed ChIP and Luciferase assay to validate Snail1 binds to different E-box motifs of our candidates. Additionally, we analyzed the ability to prevent the differentiation to adipocytes of the 3T3-L1 cells using siRNAs. This work provided insight into novel proteins with potential roles in the regulation of differentiation to adipocytes of the 3T3-L1 and mMSC cells as Nr2F6, ASC-1, Prrx1 or Cbx6 controlled by Snail1.

Dataset Information

Mapping the spatial proteome of metastatic cells in colorectal cancer

Publications

Mapping the Spatial Proteome of Metastatic Cells in Colorectal Cancer.

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