University of Vermont COBRE (P20 RR016435) 
"Center for Neuroscience Excellence"

Pilot Project 3: "Regulation of Aspartoacylase in Glioma: Potential Role as a Tumor Suppressor"
Investigator: Diane Jaworski, Ph.D.

Despite multidisciplinary approaches, the overall median survival time for patients with glioblastoma multiforme (GBM), the most common malignant primary adult brain tumor, is ~ 1 year after diagnosis. One treat¬ment chal¬lenge is the insidious propensity of glioma cells to aggressively infiltrate, making surgical resection palliative rather than curative. One of the most frequent routes of invasion is along white matter tracts, even though central nervous system (CNS) myelin is very inhibi¬tory for cell migration. Another treatment challenge is recurrence due to the persistence of chemo- and radiation resistant glioma stem cells (GSCs). It is well accepted that the extracellular microenviron¬ment is a key contributor to gliomagenesis and much attention has focused on the role of proteases. However, the poor outcomes of the matrix metallo¬proteinase (MMP) inhibitor clinical trials suggest a significant contribution of other protease families to tumor progression.

 Using TaqMan low density array (TLDA) microfluidic cards containing 380 degradome genes, the protease signature of normal brain was compared to metastatic and primary brain tumors. Only one gene, aminoacylase 3 (ACY3), was exclu¬sively up-regulated in astrocytoma relative to normal brain. Intriguingly, only two genes were significantly down-regulated exclusively in astrocytoma, one of which is aspartoacylase (ASPA, aminoacylase 2). ASPA is strongly ex¬pressed by oligodendrocytes throughout white matter pathways. Loss of APSA, the primary brain enzyme to metabolize N-acetyl-aspartate (NAA), is associated with Canavan Dis¬ease (CD); a leukodystrophy characterized by NAA accumula¬tion and white matter degeneration, suggesting ASPA plays a role in myelin formation/maintenance. We show that ASPA is more abundantly expressed in nestin-positive progenitors than GFAP-positive U373 astrocytoma cells. Further¬more, we provide provoca¬tive prelimi¬nary data that ASPA re-expression to physiological levels in human oligodendroglioma (HOG) cells is associ¬ated with decreased cell growth and migration.

 The A1 R21 application that this COBRE Pilot Project is based upon sought to characterize the inverse relationship of ACY3 and ASPA in glioma in vivo and in vitro. The summary statement clearly indicated that the descriptive experiments in Aim 1 (i.e., immunochemical characterization in normal brain and glioma as well as normal astrocytes and glioma cell lines in vitro) reduced enthusiasm for the proposal. Using VCC/LCCRO Pilot Project funds, we have not only completed Aim 1, but also generated the tet-inducible lines and show reduced cell growth and migration upon restoration of ASPA expression. Since Aim 1 was completed, a new Aim 1 was proposed in the A2 submission. Based on the ASPA expression in GSCs, the entire A2 proposal focuses only on ASPA and it seeks to test the hypothesis that ASPA regulates stem cell quiescence. Ergo, ASPA down-regulation in glioma induces loss of cell cycle arrest and, via its traditional role in NAA metabolism, increased invasion due to disrupted myelin integrity.