Dataset Information

Cell-specific analysis of tumour-endothelial contact-intiated signalling

ABSTRACT: Tumour cell extravasation is a key event for metastatic colonisation1. However, little is known about the signalling pathways that govern tumour cell adhesion to the endothelium and subsequent transmigration through the blood vessel wall. Here, we combine Stable Isotope Labelling by Amino acids in Cell culture (SILAC) and global phosphoproteomic analysis to identify cell-specific signalling pathways regulated between interacting tumour and endothelial cells. We identified over 2,000 unique phosphorylation sites, where 77 were specifically regulated in the tumour cells and 43 in the endothelial cells.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Breast Cancer

SUBMITTER: Marie Locard-Paulet

LAB HEAD: Claus Jorgensen

PROVIDER: PXD001558 | Pride | 2016-02-12

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20120229_MLP_333188_ps108TiO2.raw	Raw
	20120229_MLP_333188_ps108TiO2_V5.csv	Csv
	20120229_MLP_333188_ps109TiO2.raw	Raw
	20120229_MLP_333188_ps109TiO2_V5.csv	Csv
	20120229_MLP_333188_ps110ATiO2.raw	Raw

Items per page:

1 - 5 of 91

Publications

Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration.

Locard-Paulet Marie M Lim Lindsay L Veluscek Giulia G McMahon Kelly K Sinclair John J van Weverwijk Antoinette A Worboys Jonathan D JD Yuan Yinyin Y Isacke Clare M CM Jørgensen Claus C

Science signaling 20160209 414

The exit of metastasizing tumor cells from the vasculature, extravasation, is regulated by their dynamic interactions with the endothelial cells that line the internal surface of vessels. To elucidate signals controlling tumor cell adhesion to the endothelium and subsequent transendothelial migration, we performed phosphoproteomic analysis to map cell-specific changes in protein phosphorylation that were triggered by contact between metastatic MDA-MB-231 breast cancer cells and endothelial cells ...[more]

PMID: 26861043

Similar Datasets

Project description:Metastasizing tumor cells exit the vascular system through dynamic interactions with endothelial cells that line the internal surface of vessels. While extravasation is a key event within the metastatic cascade, the signals regulating tumor cell adhesion to the endothelium and their subsequent transendothelial migration are poorly understood. Here, we combined Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and phosphoproteomic analysis to identify cell-specific signaling pathways regulated between interacting breast cancer cells and endothelial cells (see PRIDE repository PXD001558). Further co-culture experiments were performed alongside the phosphoproteomic analysis in order to control for the protein quantity. These are presented here. Using SILAC, cell-specific labels were introduced into MDA-MB-231-LM2 cells (Human Breast cancer) and HUVECs (Human endothelial cells) to ensure each cell type had a distinct and traceable phosphoproteome when tumor and endothelial cells were co-cultured. To probe regulatory signaling events triggered in cancer cells following contact with endothelial cells, we labeled LM2 cells with medium or heavy isotopomers of arginine and lysine (Arg+6 Da, Lys+4 Da and Arg+10 Da, Lys+8 Da respectively). Heavy-labeled LM2 cells were collected by enzyme-free cell dissociation buffer, thereby preserving membrane proteins and adhesion receptors, and seeded onto a monolayer of light-labeled (Arg+0 Da, Lys+0 Da) HUVECs. Following 15 min of co-culture, non-adherent LM2 cells were gently removed and cancer cells that had attached to the endothelial layer were lysed together with the HUVECs. In parallel, medium-labeled LM2 cells were collected under the same conditions and maintained as suspension cells in monoculture to represent circulating tumor cells prior to any contact with the endothelium. These were then added to the harvested LM2-HUVEC co-culture in a 1:1 ratio of heavy:medium-labeled cells to provide a point of reference. Based on the SILAC labeling of the different cell populations, light-labeled peptides were assigned to HUVECs, medium-labeled peptides to monocultured LM2 cells in suspension, and heavy-labeled peptides to LM2 cells that had made contact with HUVECs. As such, the heavy/medium ratio for each peptide was used to quantify phosphorylation-dependent signaling changes occurring specifically in the LM2 cells upon contact with HUVECs. Conversely, to elucidate signaling events in HUVECs that were initiated by contacting cancer cells, light-labeled LM2 cells were seeded on top of a confluent monolayer of heavy-labeled HUVECs, while medium-labeled HUVECs were maintained in monoculture. Following 15 min of co-culture, the unattached LM2 cells were removed. Cells were lysed, mixed in a 1:1 ratio of heavy:medium HUVECs, followed by membrane fractionation and phosphoproteomic analysis as described above. A variation in phospho-peptide quantity could be due to the experimental difficulties in mixing heavy and medium labels in a 1:1 ratio or a quick regulation of the protein quantity through modification of its expression/degradation balance. Thus, we performed relative quantification of non-phosphorylated peptides in parallel with the phosphoproteomic analysis to account for changes in total protein abundance (data presented here) or correct for experimental bias. Cytoplasmic fractions were analyzed by LC-MS/MS before TiO2 enrichment and their median log2(H/M) were used for normalization of the phosphoproteomic dataset. In order to increase the number of proteins quantified in the LM2 cells, we performed a supplementary co-culture experiment and fractionated the peptides by SDS-PAGE. In order to get a confident relative quantification of Ephrin type-A receptor 2 (EPHA2), we performed 2 independent experiments followed by EPHA2 IP and LC-MSMS.

Project description:In vertebrates, non-lens bg-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.A naturally existing 72-kDa complex of non-lens bg-crystallin (a-subunit) and trefoil factor (b-subunit), named bg-CAT, was identified from frog Bombina maxima skin secretions. Its a-subunit and b-subunit (containing three trefoil factor domains), with a non-covalently linked form of ab2, show significant sequence homology to ep37 proteins, a group of non-lens bg-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. The bg-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of bg-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC50 10 nM) and apoptosis. The bg-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membraneï®. However, what exactly target of bg-CAT act on HUVECs nuclear is still unknown. Primary cultured HUVECs treated with bg-CAT (25 nM, 2 h) were selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the genome wide level of significant differential gene expression induced by bg-CAT on HUVECs in order to get clues about bg-CAT action mechanisms. These findings illustrate novel cellular functions of non-lens bg-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals. Experiment Overall Design: We used microarrays to analysis genome wide level of significant differential gene expression induced by bg-CAT on HUVECs.Four independent biological replicates of HUVECs treated with bg-CAT (25 nM, 2 h) were compared with normal control by SAM. The significant differential expression of 123 genes (fold changeâ¥3, q value =0, FDR (false discovery rate)=0). 121 genes were up-regulated, among which members of nuclear receptor subfamily 4 (NR4A) were the most prominent. Only 2 genes were down-regulated (including collagen type I) (Supporting information Fig. S1 and Table S1 in accompanying publication). Members of NR4A exhibit differential roles in determining cell growth or cell death depending on its location in the nucleus or mitochondria. NR4A1 (also called Nur77, TR3) mediates cell apoptosis through translocation from nucleus to mitochondria and induction of cytochrome c releasing . Significant activation of NR4A members suggests these orphan nuclear receptors may be involved in the mediation of bg-CAT biological functions, like proapoptotic action of the protein at high concentrations. Up-regulated expression of a number of matrix metalloproteinases by bg-CAT could facilitate cell migration and detachment. The bg-CAT also induced the expression of a number of cytokines, including interleukin 1, interleukin 8 and interleukin 6, as well as various chemokines in HUVECs. These data provide clues for study in detail of molecular pathways through which bg-CAT exerts its biological functions.