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.
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.
From the ‡Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsløws Vej 25.3, DK-5000 Odense C, Denmark.;
Rikke R
RR
Lund
From the ‡Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsløws Vej 25.3, DK-5000 Odense C, Denmark.;
Rikke
R
Leth-Larsen
§Clinic of Pathological Anatomy and Cytology, Sydvestjysk Hospital, Finsensgade 35, DK-6700 Esbjerg, Denmark.;
Tina Di
TD
Caterino
From the ‡Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsløws Vej 25.3, DK-5000 Odense C, Denmark.;
Mikkel G
MG
Terp
From the ‡Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsløws Vej 25.3, DK-5000 Odense C, Denmark.;
Jeanette
J
Nissen
¶Department of Pathology, Slagelse Hospital, Ingemannsvej 18, DK-4200 Slagelse, Denmark.;
Anne-Vibeke
AV
Lænkholm
‖Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark;
Ole N
ON
Jensen
From the ‡Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsløws Vej 25.3, DK-5000 Odense C, Denmark.; **Department of Oncology, Odense University Hospital, Søndre Boulevard 29, DK-5000 Odense C, Denmark. hditzel@health.sdu.dk.
Henrik J
HJ
Ditzel
D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics* / Q000401:mortality / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D042966:Cytochrome-B(5) Reductase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000080864:Lactate Dehydrogenase 5; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D000077296:Niemann-Pick C1 Protein; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000172:deficiency / Q000235:genetics; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D016019:Survival Analysis; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism
D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D042966:Cytochrome-B(5) Reductase; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5; D008562:Membrane Glycoproteins; D000077296:Niemann-Pick C1 Protein; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D017475:Receptors, Nerve Growth Factor; D016212:Transforming Growth Factor beta; C497442:HIF1A protein, human; C106881:NPC1 protein, human; C583417:CYB5R3 protein, human; C508053:ERBB2 protein, human