Identification and Analysis of Signaling Networks Potentially Involved in Breast Carcinoma Metastasis to the Brain

Roswell Park Cancer Institute, United States of America
PLoS ONE (Impact Factor: 3.23). 07/2011; 6(7):e21977. DOI: 10.1371/journal.pone.0021977
Source: PubMed


Brain is a common site of breast cancer metastasis associated with significant neurologic morbidity, decreased quality of life, and greatly shortened survival. However, the molecular and cellular mechanisms underpinning brain colonization by breast carcinoma cells are poorly understood. Here, we used 2D-DIGE (Difference in Gel Electrophoresis) proteomic analysis followed by LC-tandem mass spectrometry to identify the proteins differentially expressed in brain-targeting breast carcinoma cells (MB231-Br) compared with parental MDA-MB-231 cell line. Between the two cell lines, we identified 12 proteins consistently exhibiting greater than 2-fold (p<0.05) difference in expression, which were associated by the Ingenuity Pathway Analysis (IPA) with two major signaling networks involving TNFα/TGFβ-, NFκB-, HSP-70-, TP53-, and IFNγ-associated pathways. Remarkably, highly related networks were revealed by the IPA analysis of a list of 19 brain-metastasis-associated proteins identified recently by the group of Dr. A. Sierra using MDA-MB-435-based experimental system (Martin et al., J Proteome Res 2008 7:908-20), or a 17-gene classifier associated with breast cancer brain relapse reported by the group of Dr. J. Massague based on a microarray analysis of clinically annotated breast tumors from 368 patients (Bos et al., Nature 2009 459: 1005-9). These findings, showing that different experimental systems and approaches (2D-DIGE proteomics used on brain targeting cell lines or gene expression analysis of patient samples with documented brain relapse) yield highly related signaling networks, suggest strongly that these signaling networks could be essential for a successful colonization of the brain by metastatic breast carcinoma cells.

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Available from: Vladislav V Glinsky, Oct 13, 2015
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    • "The combination of both proadhesion/migratory and pro-angiogenic gene expression signatures may dictate the organ specificity of tumor cell extravasation. The ANGPTL4 or EREG/ COX2/MMP1 and 2 signature is associated with breast cancer metastasis to the lung, while the COX2/HBEGF/ ST6GALNAC5 signature specifically mediates breast cancer cell extravasation in the brain [44] [45] [46] [47]. Moreover, efficient cancer cell transmigration through the endothelial wall may require the formation of invadopodia since it is dependent on MMP activity. "
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    • "TF-Au, PEG-Au, and untreated endothelial cell pull-down isolates resolved on NuPAGE 4-12% gradient Bis-Tris gels were stained with modified colloidal Coomassie Blue G-250 [35] and 25 individual bands were cut by hand from each lane (Fig. 1, B). In gel digestion using mass spectrometry grade trypsin gold (Promega, Madison, WI), peptide extraction, and LC-tandem mass spectrometry analyses on the Applied Biosystems-MDS-Sciex (Concorde, Ontario, Canada) 4000 Qtrap mass spectrometer were performed as previously described [36]. The tandem mass spectrometry data were processed for protein identifications using an in-house MASCOT search engine version 4.2 (Matrix Science, Boston, MA) using the Human NCBInr protein database and one missed protease cleavage site. "
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    • "Many proteins exist in several charged isoforms that can be resolved on the 2D-DIGE assay but appear as a single band on western blot. In addition, the immunoblotting results could be affected significantly by the specificities and sensitivities of the antibodies used (Li et al., 2011). Parp1 is one of the most abundant nuclear proteins involved in DNA repair and intracellular transport (Abd Elmageed et al., 2012; Schreiber et al., 2006). "
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    ABSTRACT: The Farnesoid X receptor (FXR) is a bile acid-activated transcription factor belonging to the nuclear receptor superfamily. FXR deficiency in mice results in cholestasis, metabolic disorders, and tumorigenesis in liver and intestine. FXR is known to contribute to pathogenesis by regulating gene transcription; however, changes in the post-transcriptional modification of proteins associated with FXR modulation have not been determined. In the current study, proteomic analysis of the livers of wild-type (WT) and FXR knockout (FXR-KO) mice treated with a FXR synthetic ligand or vehicle was performed. The results identified five proteins as novel FXR targets. Since FXR deficiency in mice leads to liver tumorigenesis, poly (ADP-ribose) polymerase family, member 1 (Parp1) that is important for DNA repair, was validated in the current study by quantitative real-time PCR, and 1- and 2-dimensional gel electrophoresis/western blot. The results showed that Parp1 mRNA levels were not altered by FXR genetic status or by agonist treatment. However, total Parp1 protein levels were increased in FXR-KO mice as early as 3month old. Interestingly, total Parp1 protein levels were increased in WT mice in an age-dependent manner (from 3 to 18months), but not in FXR-KO mice. Finally, activation of FXR in WT mice resulted in reduction of phosporylated Parp1 protein in the liver without affecting total Parp1 protein levels. In conclusion, this study reveals that FXR genetic status and agonist treatment affects basal levels and phosphorylation state of Parp1, respectively. These alterations, in turn, may be associated with the hepatobiliary alterations observed in FXR-KO mice and participate in FXR agonist-induced protection in the liver.
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