NFP37 SOMATIC GENE THERAPY Divulgation Abstracts phase B

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MERLO, Adrian	Kantonsspital Basel, Labor für Molek. Neuro-Onkol., Spitalstrasse 21, CH-4031 Basel tel 061 061 265 2525; fax 061 265 7138 e-mail: email
Title:	Inhibition of glioblastoma cell invasion by targeted inactivation of the focal adhesion kinase (FAK)
Co-applicants:
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DIVULGATION TEXT AT SUBMISSION (1998):

Truncated FAK proteins decrease cell motility and survival in glioblastoma cells

Graham Jones and Adrian Merlo
Molecular Neuro-Oncology, University Hospital Basel, Switzerland

Anaplastic gliomas are highly aggressive tumors arising from non-neuronal cells within the brain and which in most cases are fatal within two years of diagnosis. Several features of these tumors make therapeutic intervention particularly difficult. Firstly, the tumor cells are often resistant to both radio- and chemotherapy. Secondly, although successful surgical intervention followed by radiotherapy is the most important determinant of post-treatment survival, invariably, the patient will succumb to relentless tumor cell invasion. Significantly, the mobility and survival of such a cancer cell is increased by interactions between the tumor cell and a complex extracellular matrix (ECM). Just as the ECM is critical during normal processes of development involving the migration of cells, the composition of the ECM is also important in the progression of brain cancer. The cell responds to the ECM via a complex arrangement of proteins that transmit signals to the interior of the cell, promoting changes as diverse as cell shape and gene transcription. We believe a critical protein in this process is the Focal Adhesion Kinase (FAK), a non-receptor tyrosine kinase which is recruited to those points of the cell membrane which are in contact with the ECM. Importantly, the activity of FAK can be inhibited by introducing into the cell a shortened version of this protein, thereby suggesting a gene-therapy approach to combat brain cancer. In initial experiments using cultured monolayers of glioblastoma cells we found that inhibition of FAK decreased both the mobility and survival of these cells. Leading on from these experiments, we have developed a 3-dimensional in vitro model whereby the glioma cells aggregate into tightly packed spheroids which better reflects interactions occurring within a tumor mass. Inhibition of FAK activity had a dramatic effect on the appearance of these spheroids, disrupting cell-cell and cell-matrix contacts and increasing cell death. At present, the key question for our laboratory is to confirm whether the inhibition of FAK-dependent signalling from the ECM has similar effects in different glioma cells with different genetic backgrounds. If this is the case, we could begin to consider the next objective of optimising the inhibition of FAK signalling and the specific targeting of glioblastoma cells within the brain.

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Glioblastome sind häufige bösartige Hirntumore, welche aus klonalen Zellpopulationen aufgebaut sind, die kaum auf Chemo- und Radiotherapie ansprechen, vor allem weil sich die Tumorzellen eine eigene Umgebung aufbauen, welche die zelluläre Autonomie und Widerstandsfähigkeit verstärkt. Focal adhesion kinase (FAK) ist eine Nicht-Rezeptor Tyrosin Kinase, welche den Signalfluss zwischen der Extrazellulärmatrix und dem Zellinnern an spezifischen Kontakstellen zwischen dem Zytoskelett und der Zellmembran integriert. Die Wirkung von FAK kann gestört werden durch rekombinante C-terminale Fragmente von FAK, welche die focal adhesion targeting (FAT) Domäne enthalten. In einem in vitro Invasionsmodell haben wir die mögliche therapeutische Wirkung solcher Fragmente untersucht durch stabile Transfektion von zwei Gliomzelllinien, bei welchen alle drei p53-pl6^INK4A-pl4^ARF Tumorsuppressorgene mutiert sind, nicht aber das Suppressorgen PTEN, das nur in LN-401 alteriert ist. Dabei fanden wir, dass die stabile Expression von FAT in LN-401 Tumorzellen nicht nur das invasive Verhalten unterdrückt, sondern, und dies war überraschend, auch zu einer deutlichen Apoptosereaktion führt, unabhängig von exogenen Wachstumsfaktoren. In den LN-229 Tumorzellen kann dieser Effekt nur in Abwesenheit von exogenen Wachstumsfaktoren beobachtet werden Diese ersten Experimente geben Anlass zur Hoffnung, dass durch die biologische Manipulation des Signalflusses zwischen der Tumorzellumgebung und dem Zellinnern wesentliche Tumoreigenschaften - Invasion und Apoptoseresistenz - durchbrochen werden können. Es gilt nun zu zeigen, ob dieser Effekt durch noch gezieltere Manipulationen und gleichzeitige Gabe von Zytostatika verstärkt werden kann.

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