Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

9.1. Digital Profiling of GNS Cell Lines Discussion
In order to reveal transcriptional changes that underlie glioblastoma, I per-
formed an in-depth analysis of gene expression in malignant stem cells derived
from patient tumours in relation to untransformed, karyotypically normal neu-
ral stem cells. These cell types are closely related and it has been hypothe-
sised that gliomas arise by mutations in NS cells or in glial cells that have
reacquired stem cell features [404]. We measured gene expression by high-
throughput RNA tag sequencing (Tag-seq), a method which features high sen-
sitivity and reproducibility compared to microarrays [490]. qRT-PCR valida-
tion further demonstrates that Tag-seq expression values are highly accurate.
Other cancer samples and cell lines have recently been profiled with the same
method [346,383], which should make these samples comparable to ours and
the analyses presented in this thesis. Through Tag-seq expression profiling
of normal and cancer stem cells followed by qRT-PCR validation in a wider
panel of 22 cell lines, we identified 29 genes strongly discriminating GNS from
NS cells. Some of these genes have previously been implicated in glioma, in-
cluding four with a role in adhesion and/or migration, CD9, ST6GALNAC5,
SYNM and TES [63,241,251,477], and two transcriptional regulators, FOXG1
and CEBPB. This observation is in line with the gene ontology analysis, which
revealed "Cell adhesion” and "Cell migration” as relevant biological processes
amongst our set of differentially expressed genes (Fig 7.1), and the fact that,
although infiltrative spread is a common feature of all diffuse astrocytic tu-
mours, glioblastoma is particularly notorious for its rapid invasion of neigh-
boring brain structures [301]. Activation of the TGFβ and AKT pathways
have been described as possible molecular mediators of this invasion [245,527]
and a number of other expression profiling studies have identified a subset of
tumours with elevated expression of ECM components as well as intracellular
proteins associated with cell motility [148,390]. FOXG1, which has been pro-
posed to act as an oncogene in glioblastoma by suppressing growth-inhibitory
effects of TGFβ [448], showed remarkably strong expression in all 16 GNS lines
assayed by qRT-PCR. CEBPB was recently identified as a master regulator
of a mesenchymal gene expression signature associated with poor prognosis in
glioblastoma [85]. Studies in hepatoma and pheochromocytoma cell lines have
shown that the transcription factor encoded by CEBPB (C/EBPβ) promotes
expression of DDIT3 [140], another transcriptional regulator that we found
to be up-regulated in GNS cells. DDIT3 encodes the protein CHOP, which
in turn can inhibit C/EBPβ by dimerizing with it and acting as a dominant
negative [85]. This interplay between CEBPB and DDIT3 may be relevant for
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