Heparan sulphate proteoglycans are essential for the myeloma cell growth activity of EGF-family ligands in multiple myeloma

Oncogene (2006) 25, 7180–7191 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Heparan sulphate proteoglycans are essential for the myeloma cell growth activity of EGF-family ligands in multiple myeloma K Mahtouk1,2, FW Cremer3, T Rème1,2, M Jourdan1, M Baudard4, J Moreaux1,2, G Requirand2, G Fiol2, J De Vos1,2, M Moos3, P Quittet4, H Goldschmidt3, J-F Rossi4, D Hose3 and B Klein1,2 1 INSERM, U475, Montpellier, France; 2CHU Montpellier, Institute of Research in Biotherapy, France; 3Medizinische Klinik und Poliklinik V, Universitätsklinikum Heidelberg, INF410, Heidelberg, Germany and 4Clinical Hematology Department, CHU Montpellier, France The epidermal growth factor (EGF)/EGF-receptor (ErbB1-4) family is involved in the biology of multiple myeloma (MM). In particular, ErbB-specific inhibitors induce strong apoptosis of myeloma cells (MMC) in vitro. To delineate the contribution of the 10 EGF-family ligands to the pathogenesis of MM, we have assessed their expression and biological activity. Comparing Affymetrix DNA-microarray-expression-profiles of CD138-purified plasma-cells from 65 MM-patients and 7 normal individuals to those of plasmablasts and B-cells, we found 5/10 EGF-family genes to be expressed in MMC. Neuregulin-2 and neuregulin-3 were expressed by MMC only, while neuregulin-1, amphiregulin and transforming growth factor-a were expressed by both MMC and normal plasma-cells. Using real-time polymerase chain reaction, we found HB-EGF, amphiregulin, neuregulin-1 and epiregulin to be expressed by cells from the bone marrow-environment. Only the EGF-members able to bind heparan-sulphate proteoglycans (HSPGs) – neuregulin-1, amphiregulin, HB-EGF – promote the growth of MMC. Those ligands strongly bind MMC through HSPGs. The binding and the MMC growth activity was abrogated by heparitinase, heparin or deletion of the HS-binding domain. The number of HS-binding EGF ligand molecules bound to MMC was higher than 105 molecules/cell and paralleled that of syndecan-1. Syndecan-1, the main HSPG present on MM cells, likely concentrates high levels of HS-binding-EGF-ligands at the cell membrane and facilitates ErbB-activation. Altogether, our data further identify EGF-signalling as promising target for MM-therapy. Oncogene (2006) 25, 7180–7191. doi:10.1038/sj.onc.1209699; published online 29 May 2006 Keywords: myeloma; EGF-family; neuregulin; syndecan-1; heparin Correspondence: Dr B Klein, INSERM U475, 99 Rue Puech Villa, 34197 Montpellier, France. E-mail: Received 23 December 2005; revised 13 April 2006; accepted 19 April 2006; published online 29 May 2006 Introduction Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of malignant plasma cells in the bone marrow (BM). Despite recent therapeutic advances, the disease remains incurable with a median survival of approximately 3–4 years (Kumar et al., 2003). MM cells (MMC) are dependent on several growth factors and cytokines, produced by the MMC themselves or by the BM microenvironment. Besides the well-known MMC factors, interleukin-6 (IL-6) (Kawano et al., 1988; Klein et al., 1989) and insulin-like growth factor-1 (IGF-1) (Georgii-Hemming et al., 1996; Jelinek et al., 1997; Ferlin et al., 2000), an increasing number of additional factors are being identified, providing novel therapeutic targets for myeloma (Klein et al., 2003). The epidermal growth factor (EGF) receptor family comprises 4 members – ErbB1 (EGFR), ErbB2, ErbB3 and ErbB4 – that are involved in the development of numerous types of human cancers. Expression and/or activation of ErbB receptors are altered in many epithelial tumors and are involved in tumor progression (Holbro et al., 2003). This has led to the development of ErbB-specific inhibitors that are now at various stages of clinical development (Hynes and Lane, 2005). In MM, we have previously demonstrated that MMC express ErbB receptors, and that their activation is required for in vitro survival of MMC in a majority of patients. Indeed, a pan-ErbB inhibitor induced strong apoptosis of MMC cultured for 5 days with their BM environment in 71% of the patients (Mahtouk et al., 2004). When the ErbB-specific inhibitor was combined with dexamethasone or anti-IL-6 antibody, apoptosis was increased leading to an almost complete elimination of viable MMC while non-MMC were unaffected (Mahtouk et al., 2004). For two of the 10 EGF-ligands – HBEGF and AREG – we have shown that they support the growth of MMC in cooperation with IL-6 (Mahtouk et al., 2004, 2005). However, the significance of other EGF-family ligands in MM has yet not been elucidated. Ten ligands have been described for ErbB receptors. They can be subdivided in three groups according to their specificity: the first one includes EGF, amphiregulin (AREG), and transforming growth factor-a HSPG promote MM cell growth activity of EGF-ligands K Mahtouk et al 7181 (TGF-a) which bind to ErbB1/EGFR exclusively. The second one includes heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC) and epiregulin (EPR) which bind to both ErbB1/EGFR and ErbB4. The last one includes the four neuregulins (NRG1, NRG2, NRG3 and NRG4) which bind to ErbB3 and/or ErbB4 (Harris et al., 2003). ErbB2 has no ligand but is the preferred heterodimerization partner for other ErbBs (Citri et al., 2003). A common feature to 4 out of the 10 EGF-family members (HB-EGF, AREG, NRG1 and NRG2) is their ability to bind heparin and heparan sulfate proteoglycans (HSPGs) (Higashiyama et al., 1993; Johnson and Wong, 1994; Paria et al., 1999). NRG1 and NRG2 bind HSPGs through an immunoglobulin (Ig)-like domain, located terminally to the EGF-like domain (Loeb and Fischbach, 1995; Carraway et al., 1997). A hallmark of plasma cell differentiation is the expression of the syndecan-1 HSPG at a high density. Syndecan-1 is expressed on normal and malignant plasma cells (PC) (Wijdenes et al., 1996) and is now widely used to identify and purify PC (Sun et al., 1997; Costes et al., 1999). Syndecan-1 is found predominantly on epithelial cells. It is involved in several cellular processes that rely on interactions with extracellular matrix proteins, growth factors, chemokines and adhesion molecules (Rapraeger, 2000; Couchman, 2003). In myeloma, syndecan-1 has been shown to colocalize with growth factors in the uropods of MMC (Borset et al., 2000), and to promote hepatocyte growth factor (HGF) activity on MMC (Derksen et al., 2002). To delineate the contribution of all 10 EGF-family members to the pathogenesis of MM, we studied here their expression and biological activity in MMC. We found 5/10 EGF-family genes to be expressed in MMC compared to normal BMPC, plasmablastic cells (PPC) and B cells; 5/10 genes to be expressed in some human myeloma cell lines (HMCLs); and 4/10 genes to be expressed by cells from the BM-environment. Among all EGF-ligands, only those able to bind HSPGs can promote the growth (...truncated)