Syndecan-1 Is Essential for the Myeloma Cell Growth Factor Activity of EGF-Family Ligands in Multiple Myeloma.

Abstract

Aim was to assess the role of the epidermal growth factor (EGF)/EGF receptor (ErbB1 (EGFR), ErbB2, ErbB3 and ErbB4) family in the pathogenesis of multiple myeloma (MM) investigating (i) their expression profile and (ii) their functional activity. Comparing Affymetrix DNA-microarray-expression-profiles of CD138-purified plasma-cells from 65 MM patients and 7 normal individuals to those of plasmablastic- and B-cells, we found 5/10 EGF-family genes to be overexpressed in myeloma cells (MMC). Two of them -neuregulin(NRG)-2 and NRG-3- were expressed by MMC only, while other three -NRG-1, amphiregulin (AREG) and TGF-α- were expressed by both MMC and normal bone marrow (BM) plasma cells (PCs). In addition, using real-time PCR, we found 4/10 EGF-ligands -HB-EGF, AREG, NRG-1 and epiregulin- to be expressed by cells from the BM-environment. ErbB receptors were expressed by normal and malignant plasma cells but were lacking on plasmablastic- and B-cells. ErbB1 and ErbB2 were expressed by MMC and BMPC, whereas ErbB3 and ErbB4 were expressed by MMC exclusively. The growth of myeloma cell lines was promoted by EGF-ligands able to bind heparan sulfate (HS) proteoglycans (PGs), i.e. NRG-1, amphiregulin and HB-EGF, but not by other EGF-ligands, i.e. EGF, TGF-α, epiregulin and betacellulin. Proliferation induced by HS-binding EGF-ligands was completely abrogated when the growth factors where pre-incubated with heparin. A truncated form of NRG-1 that lacks the HS-binding domain but has kept the ErbB binding domain was unable to promote MMC growth. MMC could bind large levels of HS-binding EGF-ligands through syndecan-1, which is expressed on myeloma cells at a high density contrary to other 10 HSPGs -syndecan-2-4, glypican-1-6 and CD44v3-. The binding was abrogated by either pre-treatment with heparitinase (which cleaves HS chains), or pre-incubation of the growth factors with heparin. None of the EGF-ligands could bind or induce proliferation of the XG-10 myeloma cell line which doesn't express syndecan-1. Thus, EGF/EGF-receptor genes are expressed during plasma cell differentiation and malignant transformation: MMC express ErbB receptors and several HS-binding EGF-ligands. Cells from the BM-environment can also be a source of EGF-family members delivering a paracrine growth signal to MMC. Myeloma cell growth is stimulated by EGF-ligands that bind HS, via binding to syndecan-1, which likely concentrates high levels of HS-binding-EGF-ligands in the proximity of ErbB receptors at the cell membrane and therefore facilitates ErbB-activation. This accumulation of EGF-family members at cell membrane is required to promote their myeloma cell growth activity. We have previously shown that pan-ErbB inhibitors like CI-1033 induce strong apoptosis of primary MMC cultured with their bone-marrow environment. Together with these data, this further recommends EGF-signalling as promising target for MM therapy.