Inhibition of glioblastoma dispersal by the MEK inhibitor PD0325901.

BACKGROUND: Dispersal of glioblastoma (GBM) cells leads to recurrence and poor prognosis. Accordingly, molecular pathways involved in dispersal are potential therapeutic targets. The mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway is commonly dysregulated in GBM, and targeting this pathway with MEK inhibitors has proven effective in controlling tumor growth. Since this pathway also regulates ECM remodeling and actin organization - processes crucial to cell adhesion, substrate attachment, and cell motility - the aim of this study was to determine whether inhibiting this pathway could also impede dispersal. METHODS: A variety of methods were used to quantify the effects of the MEK inhibitor, PD0325901, on potential regulators of dispersal. Cohesion, stiffness and viscosity were quantified using a method based on ellipsoid relaxation after removal of a deforming external force. Attachment strength, cell motility, spheroid dispersal velocity, and 3D growth rate were quantified using previously described methods. RESULTS: We show that PD0325901 significantly increases aggregate cohesion, stiffness, and viscosity but only when tumor cells have access to high concentrations of fibronectin. Treatment also results in reorganization of actin from cortical into stress fibers, in both 2D and 3D culture. Moreover, drug treatment localized pFAK at sites of cell-substratum adhesion. Collectively, these changes resulted in increased strength of substrate attachment and decreased motility, a decrease in aggregate dispersal velocity, and in a marked decrease in growth rate of both 2D and 3D cultures. CONCLUSIONS: Inhibition of the MAPK/ERK pathway by PD0325901 may be an effective therapy for reducing dispersal and growth of GBM cells.

Dexamethasone-Mediated Activation of Fibronectin Matrix Assembly Reduces Dispersal of Primary Human Glioblastoma Cells.

Despite resection and adjuvant therapy, the 5-year survival for patients with Glioblastoma multiforme (GBM) is less than 10%. This poor outcome is largely attributed to rapid tumor growth and early dispersal of cells, factors that contribute to a high recurrence rate and poor prognosis. An understanding of the cellular and molecular machinery that drive growth and dispersal is essential if we are to impact long-term survival. Our previous studies utilizing a series of immortalized GBM cell lines established a functional causation between activation of fibronectin matrix assembly (FNMA), increased tumor cohesion, and decreased dispersal. Activation of FNMA was accomplished by treatment with Dexamethasone (Dex), a drug routinely used to treat brain tumor related edema. Here, we utilize a broad range of qualitative and quantitative assays and the use of a human GBM tissue microarray and freshly-isolated primary human GBM cells grown both as conventional 2D cultures and as 3D spheroids to explore the role of Dex and FNMA in modulating various parameters that can significantly influence tumor cell dispersal. We show that the expression and processing of fibronectin in a human GBM tissue-microarray is variable, with 90% of tumors displaying some abnormality or lack in capacity to secrete fibronectin or assemble it into a matrix. We also show that low-passage primary GBM cells vary in their capacity for FNMA and that Dex treatment reactivates this process. Activation of FNMA effectively "glues" cells together and prevents cells from detaching from the primary mass. Dex treatment also significantly increases the strength of cell-ECM adhesion and decreases motility. The combination of increased cohesion and decreased motility discourages in vitro and ex vivo dispersal. By increasing cell-cell cohesion, Dex also decreases growth rate of 3D spheroids. These effects could all be reversed by an inhibitor of FNMA and by the glucocorticoid receptor antagonist, RU-486. Our results describe a new role for Dex as a suppressor of GBM dispersal and growth.

Fibronectin matrix-mediated cohesion suppresses invasion of prostate cancer cells.

BACKGROUND: Invasion is an important early step in the metastatic cascade and is the primary cause of death of prostate cancer patients. In order to invade, cells must detach from the primary tumor. Cell-cell and cell-ECM interactions are important regulators of cohesion--a property previously demonstrated to mediate cell detachment and invasion. The studies reported here propose a novel role for α5ß1 integrin--the principle mediator of fibronectin matrix assembly (FNMA)--as an invasion suppressor of prostate cancer cells. METHODS: Using a combination of biophysical and cell biological methods, and well-characterized prostate cancer cell lines of varying invasiveness, we explore the relationship between cohesion, invasiveness, and FNMA. RESULTS: We show that cohesion is inversely proportional to invasive capacity. We also show that more invasive cells express lower levels of α5ß1 integrin and lack the capacity for FNMA. Cells were generated to over-express either wild-type α5 integrin or an integrin in which the cytoplasmic domain of α5 was replaced with that of α2. The α2 construct does not promote FNMA. We show that only wild-type α5 integrin promotes aggregate compaction, increases cohesion, and reduces invasion of the more aggressive cells, and that these effects can be blocked by the 70-kDa fibronectin fragment. CONCLUSIONS: We propose that restoring capacity for FNMA in deficient cells can increase tumor intercellular cohesion to a point that significantly reduces cell detachment and subsequent invasion. In prostate cancer, this could be of therapeutic benefit by blocking an early key step in the metastatic cascade.

Fibronectin matrix assembly suppresses dispersal of glioblastoma cells.

Glioblastoma (GBM), the most aggressive and most common form of primary brain tumor, has a median survival of 12-15 months. Surgical excision, radiation and chemotherapy are rarely curative since tumor cells broadly disperse within the brain. Preventing dispersal could be of therapeutic benefit. Previous studies have reported that increased cell-cell cohesion can markedly reduce invasion by discouraging cell detachment from the tumor mass. We have previously reported that α5ß1 integrin-fibronectin interaction is a powerful mediator of indirect cell-cell cohesion and that the process of fibronectin matrix assembly (FNMA) is crucial to establishing strong bonds between cells in 3D tumor-like spheroids. Here, we explore a potential role for FNMA in preventing dispersal of GBM cells from a tumor-like mass. Using a series of GBM-derived cell lines we developed an in vitro assay to measure the dispersal velocity of aggregates on a solid substrate. Despite their similar pathologic grade, aggregates from these lines spread at markedly different rates. Spreading velocity is inversely proportional to capacity for FNMA and restoring FNMA in GBM cells markedly reduces spreading velocity by keeping cells more connected. Blocking FNMA using the 70 KDa fibronectin fragment in FNMA-restored cells rescues spreading velocity, establishing a functional role for FNMA in mediating dispersal. Collectively, the data support a functional causation between restoration of FNMA and decreased dispersal velocity. This is a first demonstration that FNMA can play a suppressive role in GBM dispersal.

Iodine inhibits antiadhesive effect of PEG: implications for tissue engineering.

BACKGROUND: The success of a biomaterial implant may be affected by the surface chemistry's impact on protein adsorption. We have developed a series of poly(ethylene glycol) (PEG) containing, tyrosine-derived polycarbonates, which have been rendered radio-opaque by the iodination of tyrosine units in the copolymer backbone for use in resorbable biomedical implants including vascular stents and grafts. We tested the hypothesis that protein adsorption along with seeding, growth, and migration of human aortic smooth muscle cells (SMC) and human aortic endothelial cells (EC) will be modified by the presence of iodine and PEG within the polymer composition. METHODS: Thin films of these polymers were prepared for the protein-material and cell-material interaction studies. Dot blot, SDS-PAGE, and XPS were used to evaluate relative protein adsorption. Cell adhesion and growth studies were performed using an MTS assay. Cell migration was evaluated using an injury model. RESULTS: The presence of PEG attenuated protein adsorption, cell adhesion, and growth. With the subsequent incorporation of iodine, protein adsorption markedly increased while the antiadhesive effect of PEG was counteracted by iodine for EC and SMC adhesion and SMC growth. CONCLUSION: Iodine incorporation into the polymer resulted in increased protein adsorption thus counteracting the effect of PEG.

Inhibition of apoptosis prevents shear-induced detachment of endothelial cells.

BACKGROUND: Biomaterials placed into the vasculature in man fail to develop an endothelial lining. Attempts to seed endothelial cells (ECs) on prosthetic vascular grafts have failed due to flow-induced detachment. The mechanism of flow-induced detachment of ECs from biomaterials is undefined. We hypothesize that endothelial detachment from biomaterials is caused by flow-induced apoptosis related to the inability of human ECs to adapt to and withstand mechanical loading. MATERIALS AND METHODS: Human aortic endothelial cells were cultured on Dacron membranes, incubated in the presence or absence of a caspase inhibitor (Z-VAD-FMK), and exposed to 0, 1, 10, 20, or 30 dynes/cm(2) of shear stress for 2, 6, 12, and 24 h in a parallel plate bioreactor. The percent of ECs detached was determined and compared with no-flow controls. Apoptosis was determined by analyzing nuclear morphology and identifying cells with caspase activity using FAM-VAD-FMK. The actin cytoskeleton of cells was visualized with fluorescein phalloidin. RESULTS: Increasing shear stress resulted in detachment of ECs from the Dacron membranes, which was associated with increased apoptosis of the residual cells adherent to the membrane determined by both nuclear morphology and caspase activity. Significantly, treatment with the caspase inhibitor Z-VAD-FMK resulted in improved EC retention following exposure to high shear stress (20 and 30 dynes/cm(2)). The majority of apoptosis and detachment were determined to occur after 6 h. CONCLUSIONS: We have demonstrated that high shear stress-induced EC detachment from Dacron is an apoptosis-dependent phenomenon that can be pharmacologically inhibited by a pan-caspase inhibitor.