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NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer.

Abstract
Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol, and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic versus nonmetastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), l-lactate dehydrogenase A (LDHA), Niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04). CYB5R3 gene knock-down using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFβ and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines was confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are available via ProteomeXchange with identifier PXD001391.
AuthorsRikke R Lund, Rikke Leth-Larsen, Tina Di Caterino, Mikkel G Terp, Jeanette Nissen, Anne-Vibeke Lænkholm, Ole N Jensen, Henrik J Ditzel
JournalMolecular & cellular proteomics : MCP (Mol Cell Proteomics) Vol. 14 Issue 11 Pg. 2988-99 (Nov 2015) ISSN: 1535-9484 [Electronic] United States
PMID26351264 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Copyright© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Chemical References
  • Biomarkers, Tumor
  • Carrier Proteins
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Intracellular Signaling Peptides and Proteins
  • Isoenzymes
  • Membrane Glycoproteins
  • NPC1 protein, human
  • Niemann-Pick C1 Protein
  • RNA, Small Interfering
  • Receptors, Estrogen
  • Receptors, Nerve Growth Factor
  • Transforming Growth Factor beta
  • L-Lactate Dehydrogenase
  • Lactate Dehydrogenase 5
  • CYB5R3 protein, human
  • Cytochrome-B(5) Reductase
  • ERBB2 protein, human
  • Receptor, ErbB-2
Topics
  • Animals
  • Biomarkers, Tumor (genetics, metabolism)
  • Breast Neoplasms (diagnosis, genetics, mortality, pathology)
  • Carrier Proteins (genetics, metabolism)
  • Cell Line, Tumor
  • Cytochrome-B(5) Reductase (antagonists & inhibitors, genetics, metabolism)
  • Female
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit (genetics, metabolism)
  • Intracellular Signaling Peptides and Proteins
  • Isoenzymes (genetics, metabolism)
  • L-Lactate Dehydrogenase (genetics, metabolism)
  • Lactate Dehydrogenase 5
  • Membrane Glycoproteins (genetics, metabolism)
  • Mice
  • Mice, SCID
  • Neoplasm Metastasis
  • Neoplasm Transplantation
  • Niemann-Pick C1 Protein
  • RNA, Small Interfering (genetics, metabolism)
  • Receptor, ErbB-2 (genetics, metabolism)
  • Receptors, Estrogen (deficiency, genetics)
  • Receptors, Nerve Growth Factor (genetics, metabolism)
  • Signal Transduction
  • Survival Analysis
  • Transforming Growth Factor beta (genetics, metabolism)

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