Extracellular Endosulfatases (Sulfs) Inhibit Myeloma Tumor Growth In Vivo.

Human sulfatases Hsulf-1 and Hsulf-2 are extracellular endosulfatases that specifically remove 6-O sulfates from heparan sulfate chains. Published studies have shown that expression of Hsulf-1 in some tumor cell lines diminishes their response to heparin-binding growth factors (e.g., FGF-2, HGF) leading to speculation that these enzymes may regulate tumor growth. To examine this possibility in vivo, the cDNA for either Hsulf-1 or Hsulf-2 was expressed in the human myeloma cell line CAG. Disaccharide composition analysis of heparan sulfate chains isolated from the surface of the transfected cells reveals that 6-O sulfation of trisulfated disaccharides is diminished by 70% and 50% on cells expressing Hsulf-1 and Hsulf-2, respectively, as compared to control cells transfected with empty vector only. When stimulated with exogenous FGF-2, downstream signaling as measured by the level of phosphorylated ERK1/2, is reduced in HSulf-1 or Hsulf-2 transfectants as compared to controls. To determine if Hsulf-1 and Hsulf-2 regulate myeloma tumor cell growth in vivo, cells were injected subcutaneously into the left flank of SCID mice. Monitoring of whole animal tumor burden by analysis of human kappa light chain present in the serum revealed that tumors formed from cells expressing either Hsulf-1 or Hsulf-2 exhibited a dramatic inhibition of tumor growth as compared to tumors formed from control cells (p<0.0004). Upon sacrifice, mean weights of primary tumors were 0.46 g (n=12), 0.18 g (n=17) and 2.44 g (n=16) for Hsulf-1, Hsulf-2 and control tumors, respectively (p<0.0001). To confirm that the endosulfatase enzymes were active in vivo, assembly of the FGF-2 ternary signaling complex (FGF-2/heparan sulfate/FGF Receptor 1) was analyzed on frozen tissue sections of the tumors. Incorporation of the FGFR1 into the complex requires the presence of 6-O sulfate groups in the endogenous heparan sulfate chains. Experiments show that FGF-2 binds equally well to the heparan sulfate present in all tumors, but the FGFR1 is not incorporated into the complex on tumors expressing either Hsulf-1 or Hsulf-2. This indicates that the Sulfs are active in vivo and that this activity alters heparin-binding growth factor signaling of the tumor cells. Additionally, we find that the elevation of Sulf expression causes a marked increase in extracellular matrix (ECM) deposition within tumors which may, along with attenuated growth factor signaling, contribute to the reduction in tumor growth. These studies indicate that dynamic regulation of heparan sulfate structure by Sulfs present within the tumor microenvironment can have a dramatic impact on the growth and progression of myeloma tumors in vivo.