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

TY - JOUR

T1 - 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

AU - Lund, Rikke R

AU - Leth-Larsen, Rikke

AU - Caterino, Tina Di

AU - Terp, Mikkel Green

AU - Nissen, Jeanette

AU - Lænkholm, Anne-Vibeke

AU - Jensen, Ole N.

AU - Ditzel, Henrik J

N1 - © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

PY - 2015/11

Y1 - 2015/11

N2 - 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), Niemannpick 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 knockdown 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, TGFα and HIFβ 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.

AB - 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), Niemannpick 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 knockdown 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, TGFα and HIFβ 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.

KW - Animals

KW - Biomarkers, Tumor

KW - Breast Neoplasms

KW - Carrier Proteins

KW - Cell Line, Tumor

KW - Cytochrome-B(5) Reductase

KW - Female

KW - Gene Expression Regulation, Neoplastic

KW - Humans

KW - Hypoxia-Inducible Factor 1, alpha Subunit

KW - Isoenzymes

KW - L-Lactate Dehydrogenase

KW - Membrane Glycoproteins

KW - Mice

KW - Mice, SCID

KW - Neoplasm Metastasis

KW - Neoplasm Transplantation

KW - RNA, Small Interfering

KW - Receptor, ErbB-2

KW - Receptors, Estrogen

KW - Receptors, Nerve Growth Factor

KW - Signal Transduction

KW - Survival Analysis

KW - Transforming Growth Factor beta

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1074/mcp.m115.050385

DO - 10.1074/mcp.m115.050385

M3 - Journal article

C2 - 26351264

SN - 1535-9476

VL - 14

SP - 2988

EP - 2999

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

IS - 11

ER -