Supplemental Figure 1. A. Western blot of purified preparations of human latent and active HPSE recombinant proteins showing distinct molecular weights, β-actin was probed as a loading control. M.W. standards were run in parallel with samples (left lane). B. Dose-dependent inhibition of heparanase (A-HPSE) endoglycosidase activity by SST0001, a glycol-split, non - anticoagulant form of heparin (50, 51). The heparanase inhibitory activity and structural details of SST0001 has been described ( 50, 51). Aliquots of purified recombinant human active HPSE were treated with various concentrations of SST0001 (0, 1, 3, and 10 μg/μl,respectively) for 2 hr. at 37{degree sign}C. HPSE activity (U/ml) was then determined using the heparan sulfate degrading enzyme assay kit{trade mark, serif} (Takara, Inc.: cat # MK412) following manufacturer's protocol. Supplemental Figure 2. Western blot probed for GEF-H1 used to generate GEF-H1 knockdowns (fig. 4A). β-actin was probed as a loading control. Supplemental Figure 3. Gated populations from FACS demonstrating the selection of transduced high GFP - expressing 231BR3 cells. Supplemental Figure 4. 231BR3 GFP - expressing cells. A. 231BR3 scrambled control cells expressing GFP. B. 231BR3 GEF-H1 shRNA (clone 1) cells expressing GFP. C. 231BR3 GEF-H1 shRNA (clone 4) cells expressing GFP. Upper panel: brightfield images. Lower panel: GFP images.
ARTICLE ABSTRACT
Heparanase is the only mammalian endoglycosidase which has been widely implicated in cancer because of its capability to degrade heparan sulfate chains of heparan sulfate proteoglycans (HSPG). Specifically, the cell surface HSPG syndecan-1 and -4 (SDC1 and SDC4) are modulators of growth factor action, and SDC4 is implicated in cell adhesion as a key member of focal adhesion complexes. We hypothesized that extracellular heparanase modulates brain metastatic breast cancer (BMBC) cell invasiveness by affecting cytoskeletal dynamics, SDC4 carboxy-terminal–associated proteins, and downstream targets. We used two independently derived human BMBC cell systems (MB-231BR and MB-231BR3), which possess distinct cellular morphologies and properties. Highly aggressive spindle-shaped 231BR3 cells changed to a round cell morphology associated with expression of the small GTPase guanine nucleotide exchange factor-H1 (GEF-H1). We showed that GEF-H1 is a new component of the SDC4 signaling complex in BMBC cells. Treatment with heparanase resulted in regulation of the SDC4/protein kinase C α axis while maintaining a constitutive GEF-H1 level. Third, GEF-H1 knockdown followed by cell exposure to heparanase caused a significant regulation of activities of Rac1 and RhoA, which are GEF-H1 targets and fundamental effectors in cell plasticity control. Fourth, L-heparanase augmented expression of β1 integrin in BMBC cells and of vascular cell adhesion molecule 1 (VCAM1; the major β1 integrin receptor) in human brain microvascular endothelial cells. Finally, using a newly developed blood–brain barrier in vitro model, we show that BMBC cell transmigration was significantly reduced in GEF-H1 knockdown cells. These findings implicate heparanase in mechanisms of cytoskeletal dynamics and in the cross-talk between tumor cells and vascular brain endothelium. They are of relevance because they elucidate molecular events in the initial steps leading to BMBC onset and capturing distinct roles of latent and active heparanase in the brain microenvironment. Mol Cancer Res; 10(6); 689–702. ©2012 AACR.
