Glioblastoma multiform (GBM) is the most common and most malignant primary brain tumor. We have reported that the expression of PAX6 is decreased in most GBM patient samples when compared to adjacent normal tissue and anaplastic astrocytomas (AAs). Lower PAX6 expression in tumor tissue is an indication of an unfavorable prognosis for patients with malignant astrocytic gliomas (Zhou et. al. Clin. Can. Res. 9:3369-75). PAX6 encodes a transcription factor critical in the development of the eye and central nervous system. In contrast to the decreased expression of PAX6 in GBMs, we observed an increase in the expression of PAX6 in the GBM cell line U251 after introducing a normal chromosome 10. Loss of heterozygosity (LOH) of chromosome 10 occurs in up to 80% of human GBM cases. Therefore, the reduced expression of PAX6 in GBMs may result from the loss of unidentified tumor suppression gene(s) in chromosome 10; however, this is not asimple side effect of LOH of chromosome 10. Overexpression of PAX6 via stable transfection of a PAX6/pRC-CMV construct in the GBM cell line U251HF suppressed anchorage-independent growth in vivo and tumor formation in vitro. This data is consistent with the hypothesis that PAX6 may have a tumor-suppressor function in GBM (Zhou et. al. J. Neuro-Oncology, in print). Microarray hybridization using Affymetrix chips revealed alterations of matrix metalloproteinase-2 (MMP2) gene expression in the suppressed U251HF PAX6-transfected cells. This data was confirmed by real-time quantitative reverse transcription PCR from cells grown in vitro and in the brain of nude mice. Moreover, there is a reverse correlation between the expression of PAX6 and MMP2 in GBMs, but not in AAs. MMP2 is a well-known protease involved in cell invasion and the malignant progression of gliomas. MMP2 was also shown to be suppressed at a functional level in gelatin zymography assays in PAX6 transfectant cells. This suppression correlates with the near abolition of the ability of all PAX6 transfectants to invade in matrigel invasion assays; however, cells expressing a dominant negative mutant form of PAX6 (PAX6-344) displayed a significant enhancement in their ability to invade in this assay. Luciferase MMP2 promoter assays have indicated that the lower expression level of MMP2 in PAX6-transfected cells is due to lower MMP2 promoter activity. In conclusion, our data indicate that PAX6 plays an important role in suppressing the formation of GBM by suppressing the expression of MMP2. Identification and functional characterization of the cis-element responsible for the suppression of the MMP2 promoter activity by PAX6 is being examined.