Heparanase-Induced GEF-H1 Signaling Regulates the Cytoskeletal Dynamics of Brain Metastatic Breast Cancer Cells

Abstract

Heparanase (HPSE) is the only mammalian endoglycosidase which has been widely implicated in cancer because of its capability to degrade heparan sulfate chains (HS) of HS proteoglycans (HSPGs). Specifically, the cell surface HSPG syndecans 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, 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 demonstrated that GEF-H1 is a new component of the SDC4 signaling complex in BMBC cells. Treatment with HPSE resulted in regulation of the SDC4/protein kinase Cα axis while maintaining a constitutive GEF-H1 level. Third, GEF-H1 knockdown followed by cell exposure to HPSE caused a significant regulation of activities of Rac1 and RhoA, which are GEF-H1 targets and fundamental effectors in cell plasticity control. Fourth, L-HPSE 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 HPSE in the brain microenvironment.

MicroRNA and Protein Profiling of Brain Metastasis Competent Cell-Derived Exosomes

“…Human brain microvascular endothelial cells (HBMEC) were obtained following isolation from brain capillaries and cultured as previously described [19]. MDA-MB-231P (231P for brevity) and the brain metastatic variant MDA-MB-231BR (231BR for brevity) were provided by Dr. Patricia Steeg (The National Cancer Institute, Bethesda, MD) [19].…”

“…MDA-MB-231P (231P for brevity) and the brain metastatic variant MDA-MB-231BR (231BR for brevity) were provided by Dr. Patricia Steeg (The National Cancer Institute, Bethesda, MD) [19]. CTC1P (circulating tumor cell parental) and CTC1BMSM (circulating tumor cells possessing a brain metastasis-selected markers profile) were recently established by their isolation directly from blood of a breast cancer patient [20].…”

“…Human brain microvascular endothelial cells (HBMEC) [19] were seeded on a 12-wells plate (2×10 5 cells/well) and non-BM cell lines (1×10 6 cells) were transduced with CellLight® Tubulin-RFP (Life Technologies). Following 16 hr incubation, cells (2.5×10 5 ) of non-BM cell lines with or without exosomes from their corresponding BM derivatives were plated over the HBMEC monolayer to evaluate the differential adhesive potential.…”

Insights into the key roles of proteoglycans in breast cancer biology and translational medicine

“…Heparanase plays important roles in breast cancer metastasis to the brain, an event that signals an exceptionally poor prognosis for the patient. Heparanase was found to regulate cytoskeletal dynamics of breast cancer cells and to mediate cross-talk between tumor and brain endothelial cells that together promote metastasis to the brain [268]. Stable expression of miR-1258 in metastatic cells inhibited heparanase expression and activity and diminished experimental metastasis to brain in vivo [269].…”

Brain Metastases from Colorectal Cancer: Microenvironment and Molecular Mechanisms

“…Heparanase mediates the expression and subcellular localization of guanine nucleotide exchange factor-H1 (GEF-H1), a component of a syndecan signaling complex, thus mediates the cross-talk between tumor cells and brain endothelia and regulates the cytoskeletal dynamics of brain metastatic melanoma and breast cancer cells [73,74]. The expression level of heparanase is a significant independent risk factor for hematogenous metastasis in CRC [75].…”