Heparanase Promotes Myeloma Metastasis By Inducing EMT-like Features in Both Myeloma Cells and Endothelial Cells

Background. Bone metastases occur in 80% of patients with multiple myeloma (MM) and are a major cause of patient mortality. We previously demonstrated that MM cell-derived heparanase (HPSE), an enzyme that cleaves heparan sulfate chains, is a master regulator of MM metastasis to bone. However the mechanism by which HPSE promotes MM cell metastasis remains unclear. Recently, the epithelial-mesenchymal-transition (EMT) of cancer cells, a process which gives epithelial cells the features of mesenchymal cells, was shown to play a critical role in the metastases of solid tumors. This process enables tumor cells to lose cell-cell and cell-extracellular matrix (ECM) adhesion, and acquire motility and invasiveness, thereby facilitating metastasis. Unlike solid tumors, MM is not an epithelial-derived malignancy. MM cells do not have cell junctions, but do adhere to and interact with the ECM. Our study has shown that MM cells express both the epithelial marker E-cadherin and the mesenchymal marker vimentin. However, the existence and potential role of EMT in MM has not been investigated. Thus, in the present study, the involvement of EMT in HPSE-induced MM bone metastasis was investigated.

Methods. To assess the involvement of EMT in HPSE-promoted MM growth and metastasis, cellular protein was isolated from HPSE-low (CAG cells transfected with empty vector) and HPSE-high (CAG cells transfected with HPSE cDNA) MM cells, as well as from wild-type CAG and RPMI 8226 MM cells treated with recombinant HPSE (rHPSE) for 48 hours. Western blotting was performed and the expression of EMT markers (Vimentin, Fibronectin and RANK) was determined. The signaling pathway(s) involved in HPSE-induced EMT were identified by culturing HPSE-high cells or RPMI8226 myeloma cells with rHPSE in the presence or absence of specific signaling pathway inhibitors and measuring the expression of EMT markers. To assess the involvement of EMT in HPSE-promoted tumor progression in vivo, HPSE-low or HPSE-high MM cells were injected subcutaneously into SCID mice. Tumor growth was determined by the measurement of human kappa level in mouse sera and bone-metastasis monitored by luciferase imaging. Tumors were harvested 6 weeks after tumor cell injection and stained for EMT marker expression. Finally, to investigate whether HPSE-high MM cells induce an EMT-like phenotype in endothelial blood vessel cells, human umbilical vein endothelial cells (HUVECs) were cultured in conditioned medium of HPSE-low or HPSE-high cells for 48 hrs, and cell lysates were analyzed for EMT marker expression.

Results. Western blots demonstrated that HPSE-high MM cells as well as CAG and RPMI 8226 MM cells treated with rHPSE expressed significantly elevated levels of the mesenchymal markers Vimentin, Fibronectin and RANK, and lower levels of the epithelial marker E-cadherin, compared to HPSE-low or the untreated cells, indicating HPSE induced EMT in MM cells. The enhanced EMT in HPSE-high or rHPSE-treated MM cells was reversed by the addition of the potent ERK inhibitor PD98059, suggesting the direct involvement of ERK signaling in HPSE-induced EMT. In vivo, tumor burden and bone metastasis was significantly enhanced in mice bearing HPSE-high tumors compared with mice bearing HPSE-low tumors. Significantly higher expression of Vimentin, Fibronectin and RANK and decreased expression of E-cadherin was revealed in HPSE-high tumors, compared with the tumors formed by HPSE-low cells. In addition, the conditioned medium from HPSE-high cells significantly increased Vimentin and decreased VE-cadherin expression in HUVECs. Therefore, the same EMT in vascular endothelial cells can be induced by soluble factors secreted from HPSE-expressing MM cells in vitro.

Conclusions. (1) HPSE promotes MM progression and metastasis by inducing EMT-like features in MM cells via stimulation of the ERK signaling pathway. (2) MM cell-derived HPSE indirectly induces EMT of vascular endothelial cells. We propose that the EMT-like features induced by HPSE in both MM cells and vascular endothelial cells equips the cells with higher mobility and invasiveness, contributing to the enhanced angiogenesis and tumor bone-metastasis commonly found in MM.