Multiple myeloma arises when malignant plasma cells invade and form multiple tumors in the bone marrow. High levels of heparanase (HPSE) correlate with poor prognosis in myeloma patients. A likely target of the enzyme is the heparan sulfate (HS) proteoglycan syndecan-1 (Sdc1, CD138), which is highly expressed on myeloma cells and contributes to poor prognosis in this disease. We find that HPSE promotes an invasive phenotype mediated by the very late antigen-4 (VLA-4, or α4β1 integrin) in myeloma cells plated on either fibronectin (FN) or vascular endothelial cell adhesion molecule-1 (VCAM-1), ligands that are prevalent in the bone marrow. The phenotype depends on vascular endothelial cell growth factor receptor-2 (VEGFR2), which is aberrantly expressed in myeloma, and is characterized by a highly protrusive lamellipodium and cell invasion. HPSE-mediated trimming of the HS on Sdc1 and subsequent matrix metalloproteinase-9-mediated shedding of the syndecan exposes a juxtamembrane site in Sdc1 that binds VEGFR2 and VLA-4, thereby coupling VEGFR2 to the integrin. Shed Sdc1 can be mimicked by recombinant Sdc1 ectodomain or by a peptide based on its binding motif, which causes VLA-4 to re-orient from the lagging edge (uropod) to the leading edge of migrating cells, couple with and activate VEGFR2. Peptides (called 'synstatins') containing only the VLA-4 or VEGFR2 binding sites competitively inhibit invasion, as they block coupling of the receptors. This mechanism is also utilized by vascular endothelial cells, in which it is also activated by HPSE, during endothelial cell tube formation. Collectively, our findings reveal for the first time the mechanism through which HPSE modulates Sdc1 function to promote both tumor cell invasion and angiogenesis, thereby driving multiple myeloma progression. The inhibitory synstatins, or inhibitors of HPSE enzyme activity, are likely to show promise as therapeutics against myeloma extravasation and spread.

Syndecan-1 (Sdc1/CD138) expression is linked to disease severity in multiple myeloma (MM), although the causal basis for this link remains unclear. Here we report that capture of the IGF1 receptor (IGF1R) by Sdc1 suppresses ASK1-dependent apoptosis in MM cells. Sdc1 binds two different fractions of IGF1R, one that is constitutively active and a second that is activated by IGF1 ligand. Notably, IGF1R kinase activity in both fractions is blocked by synstatinIGF1R (SSTNIGF1R), a peptide that inhibits IGF1R capture by Sdc1, as well as by a truncated peptide (SSTNIGF1R-T) that appears to be specific for MM cells. Mechanistically, we show that ASK1 is bound to active IGF1R and inhibited by Tyr and Ser83/Ser966 phosphorylation. When IGF1R engagement with Sdc1 is blocked by SSTNIGF1R, ASK1 becomes activated, and initiates JNK- and caspase-3-mediated apoptosis. In pharmacological tests, we find SSTNIGF1R is highly stable in human plasma and displays a half-life of 27 hr in mice, wherein it significantly reduces both the size and neovascularization of CAG myeloma tumor xenografts. Taken together, our results offer a preclinical proof of concept and mechanistic rationale for the exploration of SSTNIGF1R as an experimental therapeutic to dually attack MM tumor cell survival and tumor angiogenesis.

SummaryTransmembrane 4 L six family member 5 (TM4SF5) plays an important role in cell migration, and focal adhesion kinase (FAK) activity is essential for homeostatic and pathological migration of adherent cells. However, it is unclear how TM4SF5 signaling mediates the activation of cellular migration machinery, and how FAK is activated during cell adhesion. Here, we showed that direct and adhesiondependent binding of TM4SF5 to FAK causes a structural alteration that may release the inhibitory intramolecular interaction in FAK. In turn, this may activate FAK at the cell's leading edge, to promote migration/invasion and in vivo metastasis. TM4SF5-mediated FAK activation occurred during integrin-mediated cell adhesion. TM4SF5 was localized at the leading edge of the cells, together with FAK and actin-organizing molecules, indicating a signaling link between TM4SF5/FAK and actin reorganization machinery. Impaired interactions between TM4SF5 and FAK resulted in an attenuated FAK phosphorylation (the signaling link to actin organization machinery) and the metastatic potential. Our findings demonstrate that TM4SF5 directly binds to and activates FAK in an adhesiondependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer.

The EMT (epithelial-mesenchymal transition) is involved in fibrosis and cancer, and is regulated by different signalling pathways mediated through soluble factors, actin reorganization and transcription factor actions. Because the tetraspan (also called tetraspanin) TM4SF5 (transmembrane 4 L6 family member 5) is highly expressed in hepatocellular carcinoma and induces EMT, understanding how TM4SF5 expression in hepatocytes is regulated is important. We explored the mechanisms that induce TM4SF5 expression and whether impaired signalling pathways for TM4SF5 expression inhibit the acquisition of mesenchymal cell features, using human and mouse normal hepatocytes. We found that TGFβ1 (transforming growth factor β1)-mediated Smad activation caused TM4SF5 expression and EMT, and activation of the EGFR [EGF (epidermal growth factor) receptor] pathway. Inhibition of EGFR activity following TGFβ1 treatment abolished acquisition of EMT, suggesting a link from Smads to EGFR for TM4SF5 expression. Further, TGFβ1-mediated EGFR activation and TM4SF5 expression were abolished by EGFR suppression or extracellular EGF depletion. Smad overexpression mediated EGFR activation and TM4SF5 expression in the absence of serum, and EGFR kinase inactivation or EGF depletion abolished Smad-overexpression-induced TM4SF5 and mesenchymal cell marker expression. Inhibition of Smad, EGFR or TM4SF5 using Smad7 or small compounds also blocked TM4SF5 expression and/or EMT. These results indicate that TGFβ1- and growth factor-mediated signalling activities mediate TM4SF5 expression leading to acquisition of mesenchymal cell features, suggesting that TM4SF5 induction may be involved in the development of liver pathologies.