Abstract
BackgroundA comprehensive network-based understanding of molecular pathways abnormally altered in glioblastoma multiforme (GBM) is essential for developing effective therapeutic approaches for this deadly disease.Methodology/Principal FindingsApplying a next generation sequencing technology, massively parallel signature sequencing (MPSS), we identified a total of 4535 genes that are differentially expressed between normal brain and GBM tissue. The expression changes of three up-regulated genes, CHI3L1, CHI3L2, and FOXM1, and two down-regulated genes, neurogranin and L1CAM, were confirmed by quantitative PCR. Pathway analysis revealed that TGF- β pathway related genes were significantly up-regulated in GBM tumor samples. An integrative pathway analysis of the TGF β signaling network identified two alternative TGF−β signaling pathways mediated by SOX4 (sex determining region Y-box 4) and TGFBI (Transforming growth factor beta induced). Quantitative RT-PCR and immunohistochemistry staining demonstrated that SOX4 and TGFBI expression is elevated in GBM tissues compared with normal brain tissues at both the RNA and protein levels. In vitro functional studies confirmed that TGFBI and SOX4 expression is increased by TGF- β stimulation and decreased by a specific inhibitor of TGF- β receptor 1 kinase.Conclusions/SignificanceOur MPSS database for GBM and normal brain tissues provides a useful resource for the scientific community. The identification of non-SMAD mediated TGF−β signaling pathways acting through SOX4 and TGFBI (GENE ID:7045) in GBM indicates that these alternative pathways should be considered, in addition to the canonical SMAD mediated pathway, in the development of new therapeutic strategies targeting TGF−β signaling in GBM. Finally, the construction of an extended TGF- β signaling network with overlaid gene expression changes between GBM and normal brain extends our understanding of the biology of GBM.
Highlights
Glioblastoma multiforme (GBM), the most common type of primary brain cancer, is currently incurable and uniformly fatal
We identified 22,640 massively parallel signature sequencing (MPSS) tags that have significantly expressed tags (.3 tpm in at least one pool), representing the combined transcriptome of the normal and glioblastoma multiforme (GBM) tumor tissues (Table S1). 96% of the tags could be mapped to the human genome, of which 10.1% were repeats or mapped to multiple genomic locations
We found the CHI3L2 was over expressed in GBM tissues compared to normal brain tissues (Table S5) and confirmed it by real time quantitative PCR (Figure 2)
Summary
Glioblastoma multiforme (GBM), the most common type of primary brain cancer, is currently incurable and uniformly fatal. In the past several years, DNA microarrays have been used to identify differential gene expression among different grades of brain tumors [1], for tumor classification [2,3,4], prognosis [5,6], and screening for epigenetic changes [7,8]. Despite these advances, current DNA microarray technology has limited detection sensitivity and dynamic range [9] which limits its ability to detect changes in gene expression at low levels of expression. A comprehensive network-based understanding of molecular pathways abnormally altered in glioblastoma multiforme (GBM) is essential for developing effective therapeutic approaches for this deadly disease
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