Glioblastoma is the most malignant primary brain tumor with the average
patients surviving only one year after diagnosis, even with aggressive therapy. The
formation of numerous micro-tumors dispersed into the brain due to rapid invasion of
tumor cells, presents the primary challenge to the surgical removal of tumors and
limits the effectiveness of current treatments. This dissertation presents studies aimed
at understanding the molecular mechanisms regulating invasion of human
glioblastoma cells. Transplantation of human glioblastoma cells in the zebrafish brain
showed that the knockdown of calpain 2, a calcium-activated protease, resulted in a
three fold decrease in the tumor cell invasion. The result was further verified in the
organotypic mouse brain slices where the knockdown cells demonstrated 2-fold
decrease in the area of dispersal compared to control cells. Our data show that calpain
2 plays a role in the process of tumor cell angiogenesis. Glioblastoma cells were
transplanted into the brain of zebrafish expressing GFP in the blood vessels and we
observed that 23% of animals injected with control tumor cells demonstrated
angiogenesis. In contrast, only 9% of fish that received calpain 2 knockdown cells
showed the formation of new vessels. Consistent to the reports from human
glioblastoma patients and rodent models, we did not observe metastasis of
transplanted cells outside of the brain in the zebrafish, supporting for the use of
zebrafish as an important model for glioblastoma cell invasion studies. These results
provide evidence that calpain 2 protease activity is required for the dispersal of
glioblastoma cells in the brain microenvironment. To determine the mechanism of
calpain 2 regulation of tumor cell invasion, proteolysis of filamin by calpain 2 was
studied.
Filamin is an important actin cross-linking protein which develops orthogonal
actin networks in the periphery of the cell. In this study, we show that the expression
of filamin inhibits glioblastoma cell invasion. Hence, knocking down filamin
expression by 80% resulted in 220% increase in the invasion of glioblastoma cells
through Matrigel extracellular matrix. The regulated proteolysis of filamin is a
potential mechanism to facilitate the cyclic turnover of actin orthogonal networks
which is required for glioblastoma cell invasion. In this study, we identified a novel
mechanism that the PI3 kinase activity regulates the cleavage of filamin by calpain 2
in glioblastoma cells. Binding of a membrane phospholipid phosphatidylinositol
(3,4,5) triphosphate [PtdIns (3,4,5)-P₃] to filamin induces its proteolysis by calpain 2
after the amino acid lysine 268, removing the actin binding domain which in-turn
abolishes the actin binding ability of filamin.Keywords: Zebrafish, Invasion, Calpain, Filamin, Glioblastom
