Erythropoietin (EPO)-receptor signaling induces cell death of primary myeloma cells in vitro

Thea Kristin Våtsveen, Anne-Marit Sponaas, Erming Tian, Qing Zhang, Kristine Misund, Anders Sundan, Magne Børset, Anders Waage, Gaute Brede, Thea Kristin Våtsveen, Anne-Marit Sponaas, Erming Tian, Qing Zhang, Kristine Misund, Anders Sundan, Magne Børset, Anders Waage, Gaute Brede

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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: [Trial registration number for Total Therapy (TT) 2: NCT00083551 and TT3: NCT00081939 ].

Keywords: Bone marrow stroma cells; CD138+ cells; Co-culture; ERK-1/2; Erythropoietin; Erythropoietin-receptor; JAK-2; Multiple myeloma; Survival.

Figures

Fig. 1
Fig. 1
The EPOR is expressed both at mRNA and protein levels in primary myeloma cells and cell lines. a Quantitative real-time PCR for EPOR in 36 patient samples (black) and seven HMCLs (gray). Relative quantification (RQ) was calculated using the ΔΔCt method with GAPDH as housekeeping gene. CAG HMCL was set to 1. Error bars indicate standard deviation of triplicates for each sample. b, c Flow cytometry was used to detect surface EPOR levels in myeloma cell lines and in primary myeloma samples. The data are Arcsinh transformed showing the Archsinh value of medians, and negative OH-2 is used in the first row for comparison for the cell lines
Fig. 2
Fig. 2
RhEPO-dependent reduction in cell proliferation and viability is counteracted by α-EPOR antibodies but not by stromal cells. Primary myeloma cells from three patients were treated with rhEPO as indicated and viability and proliferation were analysed. a Viability was measured by annexinV-FITC and PI staining with flow cytometry after 48 h treatment. Error bars represent variations of duplicates. b Proliferation was measured by cell ATP-release (CellTiterGlo assay) after 48 h treatment. Error bars represent variations within triplicates. Untreated sample is set to 1 for comparison. c Four primary myeloma cell samples (MM-44, MM-46, MM-47 and MM-48) were treated with 1 or 2.5 U/ml rhEpo in the presence or absence of neutralizing antibodies against EPOR as indicated. Error bars represents the variations of means of duplicates of the means of four patient samples. d Four primary myeloma samples (MM-44, MM-49, MM-50 and MM-51) were cultured with bone marrow stroma cells and 1 U/ml rhEPO. The bone marrow stroma cells did not protect the primary myeloma cells against the effect of 1 U/ml rhEPO. Error bars represents the variations of means of duplicates of means of four patient samples
Fig. 3
Fig. 3
EPOR signaling in myeloma cells. a Knockdown of EPOR in INA-6 reduces p-ERK-1/2 and p-JAK-2 after rhEPO treatment (10 U/ml, 5 min). b Immunoblotting shows specific knockdown of EPOR in INA-6 cells using siRNA directed against EPOR mRNA. c Immunoblotting of four different primary myeloma cells after rhEPO treatment (10 U/ml, 5 min) shows an increase in both p-ERK1/2 and p-JAK-2 in the patient samples
Fig. 4
Fig. 4
Prognostic relevance of EPOR expression from four independent patient cohorts. With the use of ROC optimal cutoff derived from each trial, overall survival analysis was performed on EPOR expression in the a TT2, b TT3A, c APEX trial 039 and d APEX trial 040 datasets. Low expression levels of EPOR adversely affect outcomes in all patient cohorts

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