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.
Highlights
From the ‡Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J
Intracellular proteins, that may influence the ability of cancer cells to extravasate, colonize, and establish metastasis at distant sites, we used a model system consisting of two isogenic human cancer cell lines (M-4A4 and NM-2C5) that are tumorigenic in immunodeficient mice, but only one forms metastases, M-4A4, while NM-2C5-derived primary tumors disseminate single cells to the lungs that remain dormant
IPA analysis revealed that seven of these proteins were involved in decrease of apoptosis and cell death signaling, including mitogen-activated protein kinase (MAPK) 1 (1.6-fold increase in M-4A4 cells transfected with cytochrome b5 reductase 3 (CYB5R3) siRNA versus control siRNA), BCL2-associated agonist of cell death (1.5-fold increase), and caspase 2, 6, and 8 (1.6, 1.8, and 1.6-fold decrease, respectively)(Table VI and Supplemental Fig. 2)
Summary
Cohort 1, consisting of 30 breast cancer patients who had or had not developed distant metastasis within a ten-year period following surgical removal of the primary tumor, was evaluated using staining intensity to group tumors as strongly and weakly stained using the average value as cut-off between them.
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