Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-β induced epithelial to mesenchymal transition

<p>Abstract</p> <p>Background</p> <p>TGF-β acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-β can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-β in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01.</p> <p>Results</p> <p>Preliminary experiments based on two-dimensional electrophoresis of a hydrophobic cell fraction identified only 5 differentially expressed proteins from BRI-JM01 cells. Since 3 of these proteins were glycoproteins, we next used the lectin, wheat germ agglutinin (WGA), to enrich for glycoproteins, followed by relative quantification of tryptic peptides using a label-free LC-MS based method. Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin).</p> <p>Conclusion</p> <p>Interestingly, despite the fact that TGF-β induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap. These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments. Alternatively, it is possible that the two cell lines may use different mechanisms to achieve an EMT transition.</p

Growth response of the three cell lines to recombinant human epidermal growth factor (hEGF), heregulin (HRG)-β, and transforming growth factor (TGF)-β

<p><b>Copyright information:</b></p><p>Taken from "Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition"</p><p>Breast Cancer Research 2004;6(5):R514-R530.</p><p>Published online 6 Jul 2004</p><p>PMCID:PMC549171.</p><p>Copyright © 2004 Lenferink et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> [H]thymidine incorporation measured in BRI-JM01, BRI-JM04, and BRI-JM05 cell lines after 24 hours of exposure to serial dilutions of hEGF, HRG-β, or TGF-β. Experiments were conducted in quadruplicate and repeated three times with similar results (for details see text). Western blot analysis of whole cell lysates (20 μg total protein/lane) evaluating ErbB-1, ErbB-2, ErbB-3, and ErbB-4 expression. Additional ErbB-2/Neu immunoprecipitations showed this receptor to be highly phosphorylated in the BRI-JM04 and BRI-JM05 cells. Affinity cross-link labeling of the TGF-β type I, II, III receptors expressed on the BRI-JM01, BRI-JM04 and BRI-JM05 cells using [I]TGF-βin the absence (-) or presence (+) of 100× excess of unlabeled TGF-β. Enzymatic deglycosylation using both heparinitase I/II/III and chondroitinase ABC collapses the diffusely migrating TGF-β type III receptor band to a core protein (approximately 115 kDa, indicated as TβRIII**) labeled with either [I]TGF-βor [I]TGF-β. TGF-β responsive Mv1Lu cells served as a positive control in the TGF-β growth assay and the cell surface cross-link affinity labeling studies. RT-PCR analysis of BRI-JM01 mRNA using two different primer sets (set 1, set 2) confirmed the presence of TGF-β type III receptor transcripts in the BRI-JM01 cells. TGF-β type III receptor-expressing BRI-JM05 cells (part e) were used as a positive control for TGF-β type III receptor expression, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. Experiments were repeated twice with similar results

Transforming growth factor (TGF)-βinduces an epithelial-to-mesenchymal transition (EMT) in the BRI-JM01 cell line

<p><b>Copyright information:</b></p><p>Taken from "Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition"</p><p>Breast Cancer Research 2004;6(5):R514-R530.</p><p>Published online 6 Jul 2004</p><p>PMCID:PMC549171.</p><p>Copyright © 2004 Lenferink et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Immunofluorescence microscopy demonstrating the altered localization of E-cadherin, N-cadherin and β-catenin, the rearrangement of F-actin and vimentin filaments, and the disappearance of ZO-1 that accompany the TGF-β1 induced EMT (see text for details). Flow cytometry of nonfixed live cells monitoring the presence of E-cadherin at the cell surface of BRI-JM01 cells that were exposed to TGF-βfor up to 120 hours. Nonspecific isogenic IgG (HA) and cells grown in the absence of TGF-β(CTL) were used as a negative and positive control, respectively. Western blot analysis determining the total cellular content of E-cadherin, N-cadherin and β-catenin in BRI-JM01 cells cultured for 24, 72 and 120 hours in the absence (CTL) or presence of TGF-β(see text for details). BRI-JM01 cells were treated with TGF-βfor 48 hours (BRI-JM01T) and passaged several times in the absence of this growth factor. When seeded in Matrigel these 'TGF-βprimed' BRI-JM01 cells formed larger three-dimensional structures than did the nontreated cells

Evaluation of the activated Neu and transforming growth factor-β type II receptor antisense RNA (TβRII-AS) gene expression in mouse mammary tumor virus (MMTV) transgenic mouse mammary tumors and the derived cell lines

<p><b>Copyright information:</b></p><p>Taken from "Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition"</p><p>Breast Cancer Research 2004;6(5):R514-R530.</p><p>Published online 6 Jul 2004</p><p>PMCID:PMC549171.</p><p>Copyright © 2004 Lenferink et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Activated Neu and TβRII-AS transgene expression was determined by RT-PCR in four independent MMTV/activated Neu + TβRII-AS mammary tumors (left panel), in one MMTV/activated Neu mammary tumor (right panel), and the BRI-JM01, BRI-JM04 and BRI-JM05 cell lines. First strand reactions were carried out in the presence or absence of reverse transcriptase (± RT). Genomic DNA obtained from the BRI-JM01, BRI-JM04, and BRI-JM05 cell lines was evaluated by PCR for the presence of the TβRII-AS (middle) and activated Neu (right) transgenes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as positive control (parts a and c, left panels)