Leukemia (2013) 27, 1729–1737 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu ORIGINAL ARTICLE The hypoxia target adrenomedullin is aberrantly expressed in multiple myeloma and promotes angiogenesis KA Kocemba1,4, H van Andel1,4, A de Haan-Kramer1, K Mahtouk1, R Versteeg2, MJ Kersten3, M Spaargaren1,5 and ST Pals1,5 In multiple myeloma (MM), angiogenesis is strongly correlated to disease progression and unfavorable outcome, and may be promoted by bone marrow hypoxia. Employing gene-expression profiling, we here identified the pro-angiogenic factor adrenomedullin (AM) as the most highly upregulated gene in MM cells exposed to hypoxia. Malignant plasma cells from the majority of MM patients, belonging to distinct genetic subgroups, aberrantly express AM. Already under normoxic conditions, a subset of MM highly expressed and secreted AM, which could not be further enhanced by hypoxia or cobalt chloride-induced stabilization of hypoxia-inducible factor (HIF)1a. In line with this, expression of AM did not correlate with expression of a panel of established hypoxia-/HIF1a-target genes in MM patients. We demonstrate that MM-driven promotion of endothelial cell proliferation and tube formation is augmented by inducible expression of AM and strongly repressed by inhibition of endogenous and hypoxia-induced AM activity. Together, our results demonstrate that MM cells, both in a hypoxia-dependent and -independent fashion, aberrantly express and secrete AM, which can mediate MM-induced angiogenesis. Thus, AM secretion can be a major driving force for the angiogenic switch observed during MM evolution, which renders AM a putative target for MM therapy. Leukemia (2013) 27, 1729–1737; doi:10.1038/leu.2013.76 Keywords: adrenomedullin; hypoxia; myeloma; angiogenesis INTRODUCTION MGUS, smoldering MM and active MM,10 it has been suggested Multiple myeloma (MM) is a neoplasm characterized by expansion that the angiogenic switch could also be the consequence of of malignant plasma cells in the bone marrow. The transition of a increasing tumor burden, rather than that of aberrant expression of normal plasma cell to a fully transformed, aggressive myeloma pro-angiogenic factors alone. In line with this notion, a study by Hose 11 cell is a multistep process, which requires the acquisition of et al. revealed that even normal bone marrow plasma cells (BMPC) chromosomal translocations and mutations in multiple genes. have significant pro-angiogenic properties. On the other hand, Most of this evolution takes place in the bone marrow (BM), chronic hypoxia may also have an important role in BM angiogenesis 12–14 indicating that the interaction with the BM microenvironment in MM. This is suggested by studies demonstrating stabilization has a critical role in the pathogenesis of MM.1,2 In the MM- and nuclear localization of the HIF1a protein in malignant plasma 12,13,15,16 infiltrated BM, aberrant neovascularization (angiogenesis) is cells. Indeed, by employing gene-expression profiling, Colla 13 almost invariably present, and is associated with endothelial et al. demonstrated that hypoxia affects the transcriptional and activation, increased capillary permeability and hyperperfusion.3,4 angiogenic profiles of myeloma cells, leading to increased expression Importantly, BM angiogenesis in MM parallels disease progression of VEGFA and IL-8 among other pro-angiogenic factors. Interestingly, and is correlated with poor event-free and overall survival,5,6 HIF1a protein stabilization and activity in MM cells may also occur while after successful treatment, microvessel density returns to under normoxic conditions and can, in collaboration with c-MYC, 15 normal.7,8 induce VEGFA-mediated angiogenesis. Together, these findings The pathogenesis of MM-induced angiogenesis has not yet suggest a central role for the hypoxia-HIF1a axis in MM-related BM been fully elucidated. It is driven by genetic alterations in the MM angiogenesis. cells resulting in an imbalance between the production of In the present study, we further explored this possibility by pro- and anti-angiogenic factors by myeloma cells and the studying the transcriptional response of MM cells to hypoxia, microenvironment. This is reflected by elevated levels of pro- using gene-expression microarrays. Interestingly, we identified the angiogenic factors, including VEGFA, bFGF, and HGF, in the BM pro-angiogenic factor adrenomedullin (AM) as the most highly plasma and peripheral blood of MM patients.9 This imbalance hypoxia-induced gene in MM cells. In primary myelomas, AM results in an ‘angiogenic switch’, which takes place on the verge of expression was found to be increased during disease progression progression of monoclonal gammopathy of undetermined from MGUS to MM. In addition, endogenous, ectopically expressed significance (MGUS) to active MM. As some of the important and hypoxia-triggered AM secretion by primary MMs and MM angiogenic factors secreted by myeloma cells, including VEGFA cell lines enhanced angiogenesis. Of note, several MM cell lines and bFGF, are equally expressed by tumor cells isolated from and primary MMs expressed high levels of AM under normoxic 1Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 2Department of Human Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands and 3Department of Hematology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Correspondence: Dr ST Pals, Department of Pathology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected] 4These authors contributed equally to this work. 5These authors share last authorship. Received 8 October 2012; revised 9 February 2013; accepted 28 February 2013; accepted article preview online 12 March 2013; advance online publication, 12 April 2013 Adrenomedullin mediates myeloma-induced angiogenesis KA Kocemba et al 1730 conditions, suggesting regulation independent of the hypoxia- MM cells (at a concentration of 106 cells/ml in RPMI medium containing 10% HIF1a axis. Taken together, our results identify AM as a potential FCS)for48hwithPMAorTNFa (100 ng/ml). The treatment with cobalt driver of the angiogenic switch and promising therapeutic chloride (CoCl2) was performed by culturing MM cells (at a concentration of 6 target in MM. 10 cells/ml in RPMI medium containing 10% FCS) for 24 h with the final concentration of CoCl2 (100 uM). HUVECs were prepared from human umbilical cord veins as described 20 MATERIALS AND METHODS previously. The adherent endothelial cells (culture flasks were coated with 1% gelatin) were maintained in RPMI medium (Invitrogen Life Preparation of complementary RNA, microarray hybridization and Technologies) containing 10% clone I serum (HyClone), 10% normal gene-expression profiling analysis human serum, 3 mg/ml of basic fibroblast growth factor (bFGF), 100 units/ RNA was extracted with the RNeasy Kit (Qiagen, Hilden, Germany) or the ml of penicillin/streptomycin and 2 mmol/l l-glutamine (complete SV-total RNA extraction kit (Promega, Fitchburg, WI, USA) and Trizol medium), and incubated at 37 1Cin5%CO2. At confluence, the cells (Invitrogen Life Technologies, Carslbad, CA, USA), in accordance with the were detached by trypsin and used in experiments before the sixth manufacturer’s instructions. Biotinylated complementary RNA was ampli- passage. fied with a double in vitro transcription, according to the Affymetrix small sample labeling protocol vII (Affymetrix, Santa Clara, CA, USA). The biotinylated complementary RNA was fragmented and hybridized to the HG- RT-PCR U133 Plus 2.0 GeneChip oligonucleotide arrays according to the manufac- Total RNA was isolated using Trizol according to the manufacturer’s turer’s instructions (Affymetrix). Fluorescence intensities were quantified and protocol (Invitrogen Life Technologies). The RNA was further purified using analyzed using the GCOS software (Affymetrix). Arrays were scaled to an isopropanol precipitation and was concentrated using the RNeasy average intensity of 100. Differentially expressed genes were identified by a MinElute Cleanup kit (Qiagen). The quantity of total RNA was measured Student’s t-test, and P-values were adjusted for multiple comparisons using using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, the Benjamini and Hochberg correction. The threshold for significance was set Wilmington, DE, USA). Five microgram of total RNA was used for cDNA 21 to a P-value of p0.05. Among those genes, those with a fold change of X2 synthesis as described previously. The PCR mixture contained: 2 ml were retained. Among the genes with a fold change X2inagiven of cDNA, 1 Â PCR Rxn buffer (Invitrogen Life Technologies), 0.2 mmol/l population, those with 100% absent call in this population were considered dNTP, 2 mmol/l MgCl2, 0.2 mmol/l of each primer, and 1 U platinum not to be biologically relevant and were removed. The call (‘present’ or Taq polymerase (Invitrogen Life Technologies). PCR conditions were: ‘absent’) is determined by Affymetrix GCOS software and indicates whether a denaturing at 95 1C for 5 min, followed by 30 cycles of 30 s at 95 1C, 30 s at gene is reliably expressed or not. For a global analysis of AM expression, gene- 58 1C and 30 s at 72 1C. The reaction was completed for 10 min at 72 1C. expression data publically available and deposited in the NIH Gene Expression Primers used were: AM forward (50-CTCTGAGTCGTGGGAAGAGG-30);