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.

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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.", "claims": [{"id": "claim-target-result", "statement": "The paper reports that high CYB5R3 expression correlated with poor disease-free and overall survival in estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04), and CYB5R3 knock-down significantly decreased lung tumor burden; no named statistical test is specified, and the MS source data are available through ProteomeXchange under identifier PXD001391.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:26351264", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "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)."}, {"asset": "source:pmid:26351264", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "CYB5R3 gene knock-down using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice."}, {"asset": "source:pmid:26351264", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "MS data are available via ProteomeXchange with identifier PXD001391."}, {"asset": "source:pmid:26351264", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "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."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "The paper reports that high CYB5R3 expression correlated with poor disease-free and overall survival in estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04), and CYB5R3 knock-down significantly decreased lung tumor burden; no named statistical test is specified, and the MS source data are available through ProteomeXchange under identifier PXD001391.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:26351264"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_absent", "description": "The GEO series accession resolves and its sample accessions are listed. 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