Introduction: Glioblastoma Multiforme (GBM) is the most prevalent and invasively aggressive primary brain tumor. Adherens Junctional Associated Protein 1(AJAP-1) is a transmembrane protein implicated in adherens junction formation in epithelial cells through interaction with the E-Cadherin-catenin complex. We recently found AJAP-1 is under-expressed in GBM cells and primary tissue, and hypothesized it to be migration related. This study aimed to understand the effect of AJAP-1 on in-vitro migration of GBM cells in relation to chemoattractants that could modulate AJAP-1’s migratory effect.

Methods: Migratory capacity was assayed using U87 GBM cells and U87 cells transfected with AJAP-1. In a transwell migration assay, cells were loaded into wells with 8 µm pores. The pores were coated with the chemoattractants vitronectin, collagen IV, E-Cadherin, CXCL-12, or a blank control. Additionally, for an adhesion assay, cells were loaded into wells spotted with the attractants and control.

Results: The transwell assay found significantly decreased migration of U87 AJAP-1 cells compared to U87 tumor cells. The adhesion assay similarly found AJAP-1 significantly impeded neurite outgrowths for E-Cadherin and CXCL-12, and increased clustering. Both findings indicate decreased migratory capacity of U87 AJAP-1 cells. Neurite outgrowths allow cells to probe surroundings and direct subsequent migration. Increased clustering indicates cells remained strongly adhered to the well surface, unable to traverse it. Furthermore, transwell migration and clustering effects for E-cadherin were muted for U87 AJAP-1 by blocking with a specific antibody, demonstrating specificity.

Conclusions: The results of this study thus implicate AJAP-1 as a migratory inhibitor; loss of AJAP-1 as in GBM cells is implicated in their increased migratory capacity. Both blockage of E-Cadherin with an antibody and under-expression of AJAP-1 as in U87 tumor cells increased migratory capacity. Thus interaction of AJAP-1 with E-Cadherin appears to be involved in maintaining a decreased migration potential through an unknown mechanism.

Patient Care: Successful surgical resection of GBM tumors allows for only 8 months median survival time, which can be increased with concurrent radiation therapy and temozolomide chemotherapy to 12 - 14 months. The biggest impediment to treatment is the invariable characteristic of malignant glioma cells to invade deeply into surrounding normal brain parenchyma, limiting the effectiveness of surgical resection. Yet, this aspect of glioma invasion remains untargeted by current adjuvant chemobiological therapy as the molecular mechanism for glioma cell migration across a surface and invasion through a 3D extracellular matrix is unknown. This study identifies loss of AJAP-1 to be implicated in glioma migration, and suggests that the interaction of AJAP-1 and E-Cadherin plays a role in inhibiting migration. Though the exact mechanism of this interaction is yet unknown, this study identifies AJAP-1 and potentially E-Cadherin as targets for future research in better understanding glioma invasion, and how to target it in therapy.

Learning Objectives: By the conclusion of this session, participants should be able to: 1) Describe the importance of loss of AJAP-1 in allowing for increased migratory capacity of GBM tumor cells 2) Discuss, in small groups the potential role the interaction of E-Cadherin and AJAP-1 plays in inhibiting migration, and 3) Identify an effective potential target of treatment to repress glioma migration.

References: Bharti S, H.-M. H., Zickenheiner S, Zeitvogel A, Baumann R, Starzinski-Powitz A. (2004). "Novel membrane protein shrew-1 targets to cadherin-mediated junctions in polarized epithelial cells." Molecular Cell Biology 15: 397-406. Cifarelli CP, Titus B, Yeoh HK. “Cadherin-dependent adhesion of human U373 MG glioblastoma cells promotes neurite outgrowth and increases migratory capacity.” J Neurosurg. 2011 March; 114(3):663-9. Epub 2010 Apr 23. Di C, Mattox AK, Harward S, Adamson DC. “Emerging therapeutic targets and agents for glioblastoma migrating cells.” Anti-Cancer Agents in Medicinal Chemistry. 2010; 10:543-55. Lin N, D. C., Bortoff K, Fu J, Truszkowski P, Killela P, Duncan C, McLendon R, Bigner D, Gregory S, Adamson DC "Deletion or epigenetic silencing of AJAP1 on 1p36 is a frequent event in glioblastoma.”