Trim3, the Human Homolog of Drosophila Brain Tumor, Regulates Neural Differentiation, c-Myc Expression and Glioma Growth Properties
DJ Brat, G Chen, F Rahman, Y Rong, C Tucker-Burden, C Hadjipanayis, EG Van Meir. Emory University School of Medicine, Atlanta, GA
Background: Glioblastoma (GBM) is the most common malignant brain tumor. A CD133+ stem cell compartment has been described, yet its specialized properties are not fully understood. Drosophila Brain tumor (brat) directs asymmetric neuroblast division and guides neural differentiation in the developing fly brain at least partially through its suppression of Myc. Brat mutation results in a massively enlarged brain consisting of neuroblasts with neoplastic properties. We investigated the role of brat homologs in directing zebrafish neural differentiation and in human GBMs for a potential role in modulating c-Myc and growth properties.
Design: Morpholino oligos directed at the zebrafish homolog of brat, Trim3b, were injected into single cell embryos. In situ hybridization and electron microscopy were used to characterize the resulting CNS phenotype. The human homolog of brat, Trim3, was studied in GBM cell lines, neurosphere cultures and GBM specimens by RT-PCR and Western blot. Transient transfection and lenitviral infection of Trim3 expression constructs and shRNAs were used to study effects on c-Myc expression and glioma growth.
Results: Knock-down of Trim3b in zebrafish resulted in an enlarged head, reduced cell numbers and an enrichment of neuroblastic cells lacking differentiation. Using transgenic GFAP-GFP and Hu-GFP zebrafish, we found a marked reduction in the expression of these differentiation markers in Trim3b morpholino-treated fish. The stem cell marker Notch1a was slightly reduced in treated fish, consistent with lower cell numbers. The human homolog of brat, Trim3 (11p15.5), shows allelic loss in 25-30% of GBMs. We found that Trim3 mRNA and protein expression were reduced in human GBM cell lines and specimens compared to astrocytes and normal brain, respectively. Overexpression of Trim3 in human GBM cell lines led to reduced c-Myc protein and Myc target gene expression. This inverse relationship between Trim3 and c-Myc also held in GBM samples. Lentiviral infection of GBM cell lines with Trim3 expression constructs led to reduced proliferation and colony formation in soft agar.
Conclusions: We suggest that the reduced Trim3 expression noted in human GBMs results in upregulated c-Myc, which could potentially enrich the stem cell compartment and enhance growth properties.
Tuesday, March 23, 2010 1:15 PM
Platform Session: Section H, Tuesday Afternoon