title: Erythropoietin (EPO)-receptor signaling induces cell death of primary myeloma cells in vitro.
abstract: Multiple myeloma is an incurable complex disease characterized by clonal proliferation of malignant plasma cells in a hypoxic bone marrow environment. Hypoxia-dependent erythropoietin (EPO)-receptor (EPOR) signaling is central in various cancers, but the relevance of EPOR signaling in multiple myeloma cells has not yet been thoroughly investigated.
  
  
  
  Myeloma cell lines and malignant plasma cells isolated from bone marrow of myeloma patients were used in this study. Transcript levels were analysed by quantitative PCR and cell surface levels of EPOR in primary cells by flow cytometry. Knockdown of EPOR by short interfering RNA was used to show specific EPOR signaling in the myeloma cell line INA-6. Flow cytometry was used to assess viability in primary cells treated with EPO in the presence and absence of neutralizing anti-EPOR antibodies. Gene expression data for total therapy 2 (TT2), total therapy 3A (TT3A) trials and APEX 039 and 040 were retrieved from NIH GEO omnibus and EBI ArrayExpress.
  
  
  
  We show that the EPOR is expressed in myeloma cell lines and in primary myeloma cells both at the mRNA and protein level. Exposure to recombinant human EPO (rhEPO) reduced viability of INA-6 myeloma cell line and of primary myeloma cells. This effect could be partially reversed by neutralizing antibodies against EPOR. In INA-6 cells and primary myeloma cells, janus kinase 2 (JAK-2) and extracellular signal regulated kinase 1 and 2 (ERK-1/2) were phosphorylated by rhEPO treatment. Knockdown of EPOR expression in INA-6 cells reduced rhEPO-induced phospo-JAK-2 and phospho-ERK-1/2. Co-cultures of primary myeloma cells with bone marrow-derived stroma cells did not protect the myeloma cells from rhEPO-induced cell death. In four different clinical trials, survival data linked to gene expression analysis indicated that high levels of EPOR mRNA were associated with better survival.
  
  
  
  Our results demonstrate for the first time active EPOR signaling in malignant plasma cells. EPO-mediated EPOR signaling reduced the viability of myeloma cell lines and of malignant primary plasma cells in vitro. Our results encourage further studies to investigate the importance of EPO/EPOR in multiple myeloma progression and treatment.
  
  
  
  [Trial registration number for Total Therapy (TT) 2: NCT00083551 and TT3: NCT00081939 ].
authors: K.G. Jebsen Centre of Myeloma Research, Department of Cancer Research and Molecular Medicine, Faculty of Medicine, NTNU, Norwegian University of Science and Technology, Trondheim, Norway.|Present Address: Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.|Present Address: Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway.
authors: Thea Kristin
authors: TK
authors: Våtsveen
authors: K.G. Jebsen Centre of Myeloma Research, Department of Cancer Research and Molecular Medicine, Faculty of Medicine, NTNU, Norwegian University of Science and Technology, Trondheim, Norway.
authors: Anne-Marit
authors: AM
authors: Sponaas
authors: Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors: Erming
authors: E
authors: Tian
authors: Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors: Qing
authors: Q
authors: Zhang
authors: K.G. Jebsen Centre of Myeloma Research, Department of Cancer Research and Molecular Medicine, Faculty of Medicine, NTNU, Norwegian University of Science and Technology, Trondheim, Norway.
authors: Kristine
authors: K
authors: Misund
authors: K.G. Jebsen Centre of Myeloma Research, Department of Cancer Research and Molecular Medicine, Faculty of Medicine, NTNU, Norwegian University of Science and Technology, Trondheim, Norway.|Centre of Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7491, Trondheim, Norway.
authors: Anders
authors: A
authors: Sundan
authors: K.G. Jebsen Centre of Myeloma Research, Department of Cancer Research and Molecular Medicine, Faculty of Medicine, NTNU, Norwegian University of Science and Technology, Trondheim, Norway.|Department of Immunology and Transfusion Medicine, St Olavs University Hospital, Trondheim, Norway.
authors: Magne
authors: M
authors: Børset
authors: K.G. Jebsen Centre of Myeloma Research, Department of Cancer Research and Molecular Medicine, Faculty of Medicine, NTNU, Norwegian University of Science and Technology, Trondheim, Norway.|Department of Haematology, St. Olavs University Hospital, Trondheim, Norway.
authors: Anders
authors: A
authors: Waage
authors: Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, NTNU, Trondheim, Norway. gabrede@gmail.com.
authors: Gaute
authors: 0000-0002-8660-140X
authors: G
authors: Brede
mesh_terms: D001853:Bone Marrow / Q000473:pathology; D016923:Cell Death; D002470:Cell Survival / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology; D006801:Humans; D009101:Multiple Myeloma / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000097:blood; D017467:Receptors, Erythropoietin / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D014407:Tumor Cells, Cultured
chemical_list: C554270:EPO protein, human; D012333:RNA, Messenger; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D011494:Protein Kinases
keywords: Bone marrow stroma cells; CD138+ cells; Co-culture; ERK-1/2; Erythropoietin; Erythropoietin-receptor; JAK-2; Multiple myeloma; Survival