Analysis of the PAX6-Mediated Suppression Pathways in Gliomas: Downregulation of MMP2 Expression

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Glioblastoma multiform (GBM) is the most common and most malignantprimary brain tumor. We have reported that the expression ofPAX6 is decreased in most GBM patient samples when comparedto adjacent normal tissue and anaplastic astrocytomas (AAs).Lower PAX6 expression in tumor tissue is an indication of anunfavorable prognosis for patients with malignant astrocyticgliomas (Zhou et. al. Clin. Can. Res. 9:3369-75). PAX6 encodesa transcription factor critical in the development of the eyeand central nervous system. In contrast to the decreased expressionof PAX6 in GBMs, we observed an increase in the expression ofPAX6 in the GBM cell line U251 after introducing a normal chromosome10. Loss of heterozygosity (LOH) of chromosome 10 occurs inup to 80% of human GBM cases. Therefore, the reduced expressionof PAX6 in GBMs may result from the loss of unidentified tumorsuppression gene(s) in chromosome 10; however, this is not asimple side effect of LOH of chromosome 10. Overexpression ofPAX6 via stable transfection of a PAX6/pRC-CMV construct inthe GBM cell line U251HF suppressed anchorage-independent growthin vivo and tumor formation in vitro. This data is consistentwith the hypothesis that PAX6 may have a tumor-suppressor functionin GBM (Zhou et. al. J. Neuro-Oncology, in print). Microarrayhybridization using Affymetrix chips revealed alterations ofmatrix metalloproteinase-2 (MMP2) gene expression in the suppressedU251HF PAX6-transfected cells. This data was confirmed by real-timequantitative reverse transcription PCR from cells grown in vitroand in the brain of nude mice. Moreover, there is a reversecorrelation between the expression of PAX6 and MMP2 in GBMs,but not in AAs. MMP2 is a well-known protease involved in cellinvasion and the malignant progression of gliomas. MMP2 wasalso shown to be suppressed at a functional level in gelatinzymography assays in PAX6 transfectant cells. This suppressioncorrelates with the near abolition of the ability of all PAX6transfectants 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 invadein this assay. Luciferase MMP2 promoter assays have indicatedthat the lower expression level of MMP2 in PAX6-transfectedcells is due to lower MMP2 promoter activity. In conclusion,our data indicate that PAX6 plays an important role in suppressingthe formation of GBM by suppressing the expression of MMP2.Identification and functional characterization of the cis-elementresponsible for the suppression of the MMP2 promoter activityby PAX6 is being examined.


Abstract #2809 from poster presented at 96th Annual Meeting of the American Association for Cancer Research, Anaheim, CA, 2005.