J Neurooncol (2009) 92:261273

DOI 10.1007/s11060-009-9829-0

TOPIC REVIEW

The evolution and application of techniques in molecular biology

to human brain tumors: a 25 year perspective
James T. Rutka Paul Kongkham Paul Northcott Carlos Carlotti
Mustafa Guduk Hirokatsu Osawa Orlando Moreno Ho Jun Seol
Andres Restrepo Adrienne Weeks Shoichi Nagai Christian Smith

Received: 1 December 2008 / Accepted: 23 February 2009

Springer Science+Business Media, LLC. 2009

Abstract Since the establishment of the AANS/CNS

Section on Tumors in 1984, neurosurgeons have been
actively involved in basic science research of human brain
tumors that has moved the field forward considerably.
Here, we chronicle the major advances that have been
made with respect to our understanding of the concepts
guiding the biology of human malignant brain tumors.
Numerous technical advances in science, such as the
development of gene transfer techniques, the polymerase
chain reaction, the discovery of oncogenes and tumor
suppressor genes, and the refinement of approaches to
cancer cytogenetics have enabled researchers to identify
many of the non-random genetic alterations associated with
brain tumor growth, invasion, immunology, angiogenesis
and apoptosis. These data led to some astounding progress,
for example with the use of gene therapy, whereby in the
1990s several human clinical trials were conducted for
patients with brain tumors. More recently, the human
genome project has been completed providing a blueprint
for the human species. What has followed are exciting new
techniques in molecular biology such as transcriptional
profiling, single nucleotide polymorphism (SNP)-arrays,
array comparative genomic hybridization (array-CGH),
microRNA profiling, and detection of epigenetic silencing
of tumor suppressor genes. The cancer genome is now
being sequenced at break neck speed using advanced DNA
sequencing techniques. We are on the threshold of
J. T. Rutka (&)
P. Kongkham
P. Northcott
C. Carlotti

M. Guduk
H. Osawa
O. Moreno
H. J. Seol
A. Restrepo

A. Weeks
S. Nagai
C. Smith
The Arthur and Sonia Labatt Brain Tumour Research Centre,
Division of Neurosurgery, The Hospital for Sick Children, The
University of Toronto, Suite 1503, 555 University Avenue, M5G
1X8 Toronto, ON, Canada
e-mail: james.rutka@sickkids.ca

cataloguing the major genetic alterations observed in all

human brain tumors. What will follow is modeling of these
genetic alterations in systems that will allow for the
development of novel pharmacotherapeutics and translational research therapies.
Keywords Brain tumor
Molecular biology

Cytogenetics
Epigenetics
MicroRNAs

DNA sequencing

Introduction
Patients with malignant brain tumors continue to pose
tremendous challenges to their treating neurologists, neurosurgeons, radiation oncologists, and neuro-oncologists.
Twenty-five years ago, our understanding of the basic
biology of brain tumors was in its infancy. There was
arguably little progress that could be claimed then that
would impact favorably on the course of the disease for
most patients. However, in the short course of 25 years, our
knowledge base for these tumors has increased dramatically. At least some of the victories that we can now claim
for patients with malignant brain tumors arise out of a
better characterization of the signaling and cell cycle
pathways, cell surface markers, tumor microenvironment,
and genetic and epigenetic disturbances that comprise these
tumors. Here, we chronicle the formidable advances that
have occurred through the application of techniques in
molecular biology to the study of human brain tumors over
the past 25 years. It is probably fair to say that since the
creation of the AANS/CNS Section on Tumors in 1984, the
techniques used to probe the most fundamental aspects of
these tumors have advanced at a faster rate than has
ever occurred before in history. For example, whereas in

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1984 the genetic alterations in human brain tumors were

determined through conventional G-banding karyotyping
(Fig. 1), and required a skilled cytogeneticist for interpretation, today comprehensive genome wide surveys and
sequencing can be performed which sift through billions of
datapoints to arrive at the key genetic disturbances that
characterize human cancer.

A look into the past: brain tumor research

in the 1980s
In the early 1980s, streams of advancements in genetics and
molecular biology were converging to provide new tools
with which research investigators could ask questions
regarding the etiology of human brain tumors. Among these
tools were monoclonal antibodies which could be used to
detect the presence or quantity of a protein in tumor cell
lysates using Western blot analysis or in fixed and preserved
tumor specimens using immunohistochemistry. The promise of monoclonal antibodies was exciting for if brain
tumors were found to express unique antigens which were
not expressed by normal cells, then antibodies could be
modified so as to deliver a toxin, radio-isotope or cytokine
selectively to a brain tumor cell. This line of investigation
has been pursued vigorously by numerous researchers over
the years. Some of the best examples include the work by
Bigner and colleagues who determined that tenascin is
overexpressed by human malignant gliomas and against
which monoclonal antibodies were generated [1, 2]. These
antibodies, labeled with radio-isotopes, have now been used
in clinical trials in patients harboring malignant gliomas
with significant efficacy [39].
Just as the field of monoclonal antibodies and immunotherapy was developing in the early 1980s, the recognition
Fig. 1 Classic G-banding
karyotyping of a
medulloblastoma is seen in
panel (D). CGH of this tumor
reveals gains on chromosomes 5
and 7, panel (A). SKY paints are
applied to the tumor providing
color coding of each
chromosome, panels (B) and
(C). Translocations are readily
seen in the SKY metaphase
preparation of this
medulloblastoma involving
chromosomes 3 and 7; 10 and
14; and 7q and 11 q, panel (E).
From Bayani et al. [31] with
permission

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of latent cancer causing genes in the human genome, socalled oncogenes was quickly becoming appreciated. In
1976, Bishop and Varmus demonstrated that oncogenes
were defected proto-oncogenes that are found in several
species including humans [10]. Proto-oncogene activation
may be caused by point mutations, chromosomal translocations, deletions and gene amplifications [11]. Among
some of the oncogenes that were identified in human brain
tumors at that time were c-myc, N-ras, v-sis, and v-fos for
human malignant gliomas [12].
It was in the 1980s that the hypothesis that growth
factors, growth factor receptors, and their related biochemically active secondary and tertiary messages was
rapidly gaining momentum. It was hypothesized that normal cellular proliferation could be disrupted, and malignant
transformation could occur if a growth factor cascade was
altered by, for example, overexpression of a particular
growth factor or growth factor receptor [12]. Some of the
growth factor activation pathways that were studied in
human brain tumors included platelet-derived growth factor
(PDGF), epidermal growth factor (EGF), and fibroblast
growth factor (FGF). Research studies from this time led to
the discovery that human glioblastoma multiforme (GBM)
could be characterized by amplification and overexpression
of the epidermal growth factor receptor (EGFR) in as many
as 30% of cases [1315].
While investigators were beginning to analyze human
brain tumors for their expression of oncogenes, growth
factors, and growth factor receptors, a number of technical
advancements in molecular biology came forward to speed
up this process. They included the routine application of
restriction endonucleases to biological samples to reduce
large pieces of DNA into smaller, more manageable fragments; the development of the polymerase chain reaction
(PCR) to make large number copies of specific DNA

J Neurooncol (2009) 92:261273

fragments or genes for sequencing and further analysis; the

widespread use of DNA transfer technology which enabled
genes to be expressed ectopically in model systems and
which foreshadowed the development of gene therapy (see
next section); and the improvement of DNA sequence
technology to identify genes with greater reliability and
speed that are involved in human cancer. In 1988, the
National Center for Biotechnology Information (NCBI) was
founded to create a public database wherein biomedical
information and gene sequencing data are made available
for the better understanding of molecular processes affecting human health and disease, including cancer.

Harnessing the energy of molecular biology towards

human brain tumors: progress in the 1990s
One of the most exciting lines of investigation in the 1990s
relating to brain tumors was the use of gene therapy as a
means to control the growth and invasion of these tumors.
Gene therapy is defined as the use of nucleic acid transfer,
either RNA or DNA, to treat or prevent disease [16, 17].
Success in gene therapy requires an understanding of
disease pathogenesis, the ability to specifically target the
tissue of interest, and the elucidation of an effective therapeutic gene(s) in addition to an appropriate animal model to
study (reviewed in [18]). Glioblastoma was thought to be a
particularly good candidate for cancer gene therapy given
the rapid cellular proliferation associated with this tumor in
the context of normal postmitotic tissue, facilitating the use
of delivery vehicles or target molecules specific for dividing
cells. Pioneering work in gene therapy of malignant gliomas
involved direct injection of herpes simplex virus (HSV)
lacking the thymidine kinase (TK) gene [19]. Potent antitumor effects were noted in both established glioma cell
lines and short-term populations of glioma cells following
treatment with TK-negative HSV. Ram et al. [20] reported
that antitumor effects following implantation of retroviral
(RV) producer cells were observed only in small tumors
experimentally (\1.5 ml). Palu et al. [21] evaluated RV
delivery of IL-2 in combination with HSV-TK therapy in a
clinical trial. Follow-up of patients was brief but all four
patients were shown to have reduced tumor volume. In
essence, gene therapy seemed most promising at this time,
but subsequent studies alluded to some of the difficulties
with this technique including tumor targeting, enhanced
immunogenicity of constructs, and concern about systemic
spread of virus. The fact that human trials were conducted
with adequate safety and without significant complications
is a testament to the power of this technique. The interested
reader is referred to the results of a number of human gene
therapy trials that were conducted in the 1990s for patients
with malignant gliomas [2026].

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It was because of the rapid advances in techniques in

molecular biology in the 1980s that the US Department
of Energy and the National Institutes of Health launched
the human genome project (HGP) in 1990. While the
purpose of the HGP was to identify all genes in human
DNA, the knowledge derived from these data would
unquestionably help in the study of diseases, such as
cancer, where the direct DNA sequence may be perturbed. Numerous strategies then evolved which enabled
investigators to interrogate the cancer genome with more
precision than ever before. As a result, the field of
cancer cytogenetics developed to help researchers discover the main genetic aberrations that occur in human
brain tumors.
Fluorescein in situ hybridization (FISH) techniques
arose when molecular probes could be synthesized against
unique DNA sequences so as to visualize the gene
sequence in a given cell. FISH is used to identify numerical
and structural chromosomal abnormalities, detect minimal
residual disease, and to map gene locations. For example,
FISH is an excellent technique to identify losses of chromosome 1p and 19q in human oligodendroglioma [27], or
to see loss of chromosome 22q11 in atypical teratoid
rhabdoid tumors (ATRTs) [28].
Spectral karyotyping (SKY) was first reported in 1996,
and uses optical spectroscopy and chromosomal paints to
examine the orderly arrangement of all the human chromosomes. SKY can detect the translocation of a DNA fragment
as small as 1.5 million base pairs, and can identify the
chromosome of origin of various extrachromosomal fragments. SKY has been particularly useful in neuro-oncology
in tumors where translocations have been frequently identified, such as Ewings Sarcoma (ES) in children. Intracranial
ES can be diagnosed with accuracy by SKY through the
identification of a characteristic t(11;22)(q24:q12) translocation seen in 85% of tumors [29].
A variation on FISH technology is comparative genomic
hybridization (CGH) which, unlike FISH, allows for the
detection of changes of a few hundred kilobase pairs. CGH
quantitate the ratio of two differently colored fluorescence
probes in an in situ hybridization experiment. CGH has
been used to confirm, for example, that primary GBMs
exhibit a high frequency of chromosomal gains compared
to secondary GBMs [30].
In one of our studies, we combined the use of Giemsabanding, comparative genomic hybridization, and spectral
karyotyping to examine the genetic alterations in human
medulloblastoma. Here we found that the combination of
these three techniques led to the determination of the
greatest number of cytogenetic alterations in medulloblastoma including rearrangements on chromosomes 7 and
17, amplification on chromosome 2q in 30% of cases, and
loss of chromosome 10q (Fig. 1) [31].

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In the 1990s a powerful technique in molecular biology

emerged which enabled the detection of over- or underexpressed genes in brain tumors when compared to control,
non-neoplastic specimens. This technique is called cDNA
microarray or transcriptional profiling analysis, and uses
thousands of nucleic acid probes to interrogate the
expression of thousands of genes in a given sample. New
generation high-resolution arrays have now been developed
which interrogate every annotated human exon. In addition, the creation of genome tiling arrays enable the
measurement of gene expression at every 25 base pairs
across the entire genome so that even cryptic transcriptional units can be uncovered [32]. Transcriptional
profiling has now been performed on numerous brain tumor
subtypes such that novel genes and pathways have been
identified and further studied to increase our knowledge of
the origins of these tumors (Fig. 2) [3338]. Several years
ago, we used the Atlas 1.2 human cancer array platform to
determine genes that were overexpressed in medulloblastoma when compared to normal human cerebellum. Our
analysis led to the identification of 6 genesezrin, cyclin
D2, high mobility group protein 2, MAPRE1, histone
deacetylase 2 and ornithine decarboxylase 1which were
overexpressed several fold in medulloblastoma. From this
list of genes, we pursued the role of ezrin in medulloblastoma, and determined that its overexpression greatly
enhanced the metastatic potential of medulloblastoma in an
orthotopic xenograft model system [39].

The new millennium: oncogenomics, and epigenetic

alterations in human brain tumours
Recent advances in oncogenomics
The new millennium will be characterized by phenomenal
progress in cataloguing the multitude of genetic aberrations
that characterize human brain tumors. Numerous new
techniques are advancing the field of oncogenomics at
record breaking pace. Array CGH is one such technique,
and is similar to CGH. However, instead of hybridizing the
labeled probes to metaphase chromosomes which is done
in standard CGH cases, in array CGH the probes are
hybridized to microarray slides in which thousands of
fragments of DNA that span the genome have been
arrayed out [40]. Array CGH has been used to study a
wide variety of human brain tumors [4144]. Taylor et al.
[44] used array CGH and transcriptional profiling to show
that histologically similar ependymomas arising from different regions of the neuraxis had markedly different
patterns of copy-number-driven gene expression. Rossi
et al. [43] used array CGH in pediatric medulloblastoma to
show that loss of 17p and gain of 17q are the most common

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cytogenetic alterations in medulloblastoma, and that

amplifications are rare with the most common one being
the MYC gene on chromosome 8q.
One of the most significant advances in cancer genomics
has been the development of single nucleotide polymorphism (SNP) arrays. SNPs are naturally occurring variations
in DNA sequence that occur on average at one SNP for
every 200 bases of DNA, and are the most common form of
genetic variation in the human genome. Modern SNP array
platforms interrogate from 100,000 to 2,000,000 SNPs
resulting in high resolution genome coverage with median
SNP spacing of approximately 7001500 base pairs. This
resolution is more than 1,000 times better than classic
CGH. Recently, a comprehensive genomic characterization
was performed on 206 glioblastoma multiformes using
genomic copy number alterations (CNAs) on three microarray platforms including SNP-based arrays [45]. This study
showed high level amplifications of EGFR, CDK4, and
PDGFRA, and homozygous deletions of CDKN2A/B and
PTEN among many other findings. We have used SNP array
platforms to query over 250 primary medulloblastoma
samples, and have uncovered numerous target regions of
interest (Fig. 2) [46].
Beyond oncogenomics: cancer epigenetics
comes of age
Until recently, cancer researchers were focused primarily
on the discovery of tumor suppressor genes that were
inactivated by gene mutations and/or deletions. Now we
know that epigenetic factors can regulate gene expression,
and can lead to tumor suppressor gene silencing (Fig. 3).
Epigenetics refers to mechanisms controlling gene
expression in a heritable fashion, without any changes to
the gene sequence itself. Epigenetic mechanisms including
promoter methylation, histone modifications, and microRNAs (miRNAs) (see below) function to regulate normal
processes such as gene imprinting, X-chromosome inactivation, and developmental stage- and tissue-specific gene
expression (Fig. 4). Promoter methylation involves the
covalent addition of a methyl group to cytosine bases found
in CG dinucleotide-rich regions of the genome referred to
as CG islands. CG islands are found at the promoter
regions in approximately 60% of human genes. In normal
tissues, these CG islands are typically unmethylated,
whereas in cancer they may become extensively methylated (Fig. 5). Promoter methylation is generally associated
with transcriptional silencing, resulting from the inhibition
of transcription factor binding or the recruitment of transcriptional repressor molecules (Fig. 6) [47]. Histones are
protein complexes involved in packaging DNA into chromatin within the nucleus. The N-terminal tails of histone
proteins are subject to a myriad of post-translational

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Fig. 2 a Isochromosome 17q in medulloblastoma. High-resolution

SNP arrays are an efficient platform for detecting copy number
aberrations in tumour samples. Shown is the copy number output for
chromosome 17 in ten medulloblastomas, with losses depicted in blue
and gains in red. The most common structural abnormality in
medulloblastoma, isochromosome 17q, is observed in the majority of
the cases, as evidenced by the characteristic loss of 17p with

concomitant gain of 17q. b Gene expression profile of significant

genes on chromosome 17 in medulloblastoma. Oligonucleotide
expression arrays are a sensitive method for the detection of
differentially expressed genes in tumour samples. The heat map
highlights the most variable genes on chromosome 17 in a series of 7
medulloblastomas. Genes with reduced expression are shown in green
and those with elevated expression are red

modifications, including methylation and acetylation. In

general, acetylation is associated with a transcriptionally
competent euchromatin structure whereas deacetylation
results in a transcriptionally repressed heterochromatic
configuration. Histone methylation can be associated with
gene expression or silencing, depending on the specific
histone protein and lysine residue targeted by this modification. Taken together, the various histone modifications
have been referred to as the histone code [48]. Micro
RNAs are a group of small, non-coding RNAs involved in
regulating gene expression, and are discussed separately in
the next section.
Dysregulation of these epigenetic mechanisms may cause
altered gene expression (e.g., silencing of tumor suppressor
genes), contributing to the etiology of human cancer.
Abnormal promoter methylation or histone modifications

may confer a stable selective growth advantage on a cell

population, resulting in clonal expansion and tumorigenesis.
In some instances, epigenetic dysregulation may play an
early role in cancer initiation, with aberrant silencing of
epigenetic gatekeeper genes allowing for inappropriate
survival and expansion of a population of cells at risk of
acquiring additional genetic defects [49].
Over the last two decades there has been increasing
interest in the role epigenetic dysregulation plays, alongside genetic events, in the etiology of human brain tumors.
To date, the influence of epigenetic phenomena in brain
tumors has probably been most studied in malignant gliomas and pediatric medulloblastomas. Several classic tumor
suppressor genes, including PTEN, p53 and p14/ARF have
been shown to be silenced epigenetically in human gliomas
[5052]. Genome-wide epigenomic profiling of malignant

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Mutation

Deletion

CH 3

Epigenetics

CH 3

Fig. 3 Tumor suppressor genes may be silenced due to genetic

events such as mutation or deletion, or epigenetic events including
DNA methylation or histone modifications

Histone
Modifications

DNA
Methylation

GENE SILENCING

miRNA

Fig. 4 Epigenetic gene silencing. Several epigenetic mechanisms

cooperate to produce gene silencing, including DNA promoter
methylation, histone modifications, and micro-RNA (miRNA) mediated gene silencing. If gene silencing occurs at the locus of a tumor
suppressor gene, then tumor formation can occur

glioma cell lines and primary tumor samples has identified

a number of additional candidate genes targeted for aberrant silencing, including CST6, BIK, TSPYL5, BEX1, and

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BEX2 [53, 54]. Deregulation of the BMP signaling pathway resulting from promoter methylation-mediated
silencing of the BMP receptor 1B has been implicated in
glioma tumor stem cell tumorigenicity [55]. In addition to
its utility in understanding the pathogenesis of human brain
tumors, the study of epigenetic gene silencing may serve
prognostic purposes, as highlighted recently by the association between O(6)-methylguanine methyltransferase
(MGMT) promoter methylation and improved progressionfree and overall survival in malignant glioma patients
treated with oral alkylating chemotherapeutic agents [56].
Additional novel epigenetic prognostic markers are also
being explored [57].
Among pediatric brain tumors, medulloblastoma has
been the most intensely studied tumor type with respect to
the contribution of epigenetic phenomena to its etiology.
Strong evidence for promoter methylation-mediated epigenetic silencing in MB exists for HIC1 and RASSF1A,
with additional evidence available for a small number of
genes including CASP8, MGMT, GSTP1, p14, p15, p16,
and TIMP3 [58]. We recently showed that SPINT2, a
negative regulator of the HGF-Met signaling pathway, is
silenced by methylation in approximately 30% of primary
medulloblastomas [59]. The WNT signaling inhibitor,
DKK1, has been shown to be silenced secondary to inappropriate histone modifications in medulloblastoma [60].
MicroRNAs: small molecules with big function
MicroRNAs (miRNAs) belong to a recently described class
of small regulatory RNAs that coordinate important cellular processes, including proliferation and differentiation.
Since the 1993 discovery of lin-4 in C. elegans [61, 62],
thousands of miRNA sequences have been annotated in a
vast number of metazoan, plant, and viral genomes. With
695 unique miRNAs currently reported in humans [63], it
is estimated that they account for [3% of all human genes
[64]. As this inventory of known miRNAs continues to
increase, it is becoming increasingly evident that these
non-coding RNAs function to regulate a variety of cellular
processes including development, differentiation, proliferation, and apoptosis [64]. At present, miRNAs are probably
best described for their role in developmental processes,
most of which have been established by studies in C. elegans and Drosophila [65, 66]. In addition, several recent
reports have provided compelling evidence that deregulation of miRNAs contributes to the pathogenesis of human
disease, particularly cancer [6769].
Mature miRNAs consist of *22 nucleotide, singlestranded RNA molecules which function as a component of
the miRISC (miRNA-containing RNA-induced silencing
complex) to post-transcriptionally regulate target mRNAs
sharing sequence complementarity in their 30 untranslated

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Fig. 5 Detection of gene
promoter methylation by
bisulfite sequencing. Promoter
methylation status in normal
tissue versus tumor tissue.
Typically, CG islands remain
unmethylated (open circles) in
normal neural tissues. In brain
tumors, CG islands may become
extensively methylated,
resulting in abnormal gene
silencing (filled circles).
Vertical tick marks denote CG
sites within the promoter CG
island

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-500
EXON 1

Normal Brain

Brain Tumor

Unmethylated CG Site

Fig. 6 In normal tissue,

promoter CG islands are largely
unmethylated, resulting in a
transcriptionally competent
euchromatin state. When these
CG islands become methylated,
transcription is abolished, due to
the conversion to a closed
heterochromatin state, the
prevention of transcription
factor binding, or the
recruitment of transcriptional
repressors

Normal

Methylated CG Site

RNA

TF

TF

RNA Pol

Aberrant Promoter
CG Island Methylation

Brain Tumor
RNA Pol

TF
TF

Transcription
Abolished

Unmethylated CG Site

Methylated CG Site

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years, there is abundant evidence to support miRNAs acting

as oncogenes and tumor suppressors [68, 69]. An association between miRNAs and human cancer was first reported
in 2002 when it was shown that miR-15a and miR-16a map
to chromosome 13q14, a region deleted at high frequency in
chronic lymphocytic leukemia (CLL) [71]. A follow-up
study published by the same group revealed that miRNAs
are commonly located at fragile sites and other cancerassociated regions in the human genome [72]. These findings were the first indication of what has very recently
become a prevailing theme in the miRNA literature: miRNAs constitute a new class of oncogenes and tumour
suppressors.
The best characterized oncogenic miRNAs thus far
identified are components of the polycistronic mir-17-92
cluster, which maps to chromosome 13q31 and produces six
mature miRNAs [73]. This locus is recurrently amplified in
follicular lymphoma, B-cell lymphoma, and several lung,
and head and neck cancers [74, 75]. In addition, direct
evidence for this miRNA cluster acting as an oncogene has
been shown in a mouse model of B-cell lymphoma, where a
truncated portion of miR-17-92 (miR-17-19b) was capable
of cooperating with c-Myc overexpression to accelerate
lymphomagenesis [75]. Additional miRNAs have also been

regions (UTR) (Fig. 7). Target mRNAs are either degraded

or repressed translationally by the cognate miRNA,
depending on the degree of complementarity that exists
between the miRNA and the target. The short nature of
mature miRNAs and the somewhat liberal requirements for
target recognition make it possible for a single miRNA to
regulate possibly hundreds of mRNAs [63]. With the
number of predicted targets that are possible for a given
miRNA, it is not difficult to imagine how a limited number
of miRNAs may have the capacity to effectively impact
cellular phenotype. This possibility was supported in an
elegant study by Lim and colleagues [70] who demonstrated that a single miRNA could cause downregulation of
a large number of mRNAs (*100) and shift the expression
profile of miRNA-transfected cells to mirror that of the
specific tissue in which the miRNA is normally expressed.
It can be appreciated from this report how disruption in the
normal expression pattern of miRNAs might have detrimental effects on the cellular environment and, in turn,
contribute to the initiation and progression of malignancy.
The discovery of miRNAs as key post-transcriptional
regulators of the cells transcriptome has led to an intense
amount of research aimed at the characterization of these
molecules in tumorigenesis [67, 68]. Indeed, in the last few

rosha
Drosha

Pre-miRNA
nt)
re-miRNA (~80
~80 nt

Pri-miRNA
Pri-miRN

miRNA gene

Nucleus
Dicer
cer

Cytoplasm

Mature miRNA (~22 nt)

Translational repression
m7GpppG
Target mRNA

5 UTR

ORF

3 UTR

AAAAA

Imperfect
complementarity

Fig. 7 Schematic of miRNA biogenesis. miRNAs are initially

transcribed as long primary transcripts (pri-miRNAs) by RNA
polymerase II, before being processed by Drosha in the nucleus to
generate pre-miRNAs (*80 nt) which form a characteristic stemloop conformation. Pre-miRNAs are exported from the nucleus into
the cytoplasm where they are further processed by Dicer to form the

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mRNA degradation
m7GpppG
Target mRNA

5 UTR

ORF

3 UTR

AAAAA

Perfect
complementarity

mature miRNA (*22 nt). Mature miRNAs post-transcriptionally

regulate target gene expression by forming hybrid complexes with
target mRNAs in their 30 untranslated region (UTR), and subsequently
repress translation or promoting degradation of the target mRNA,
depending on the amount of complementarity that exists between the
miRNA and its target(s)

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implicated as oncogenes based upon there expression profiles in human cancer, including miR-155 which has been
reported to be aberrantly expressed in B-cell lymphoma
[76] and miR-21 which has been shown to be upregulated in
a variety of solid tumours [77, 78].
Of the miRNAs currently believed to represent bona fide
tumour suppressors, let-7 is likely the most prominent.
Expression of let-7 has been reported to be significantly
reduced in human lung cancers [79]. The significance of
this observation is underscored by the finding that the let-7
family is known to regulate Ras [80], suggesting a mechanism by which reduced let-7 expression could result in
activation of a potent oncogene.
A number of miRNAs have recently been associated with
tumours of the central nervous system. For example, miR-21
is frequently upregulated in high-grade gliomas [77, 81].
MicroRNA expression profiling showed that miR-21 was
consistently overexpressed in glioblastomas and established
glioblastoma cells lines compared to non-neoplastic control
cells derived from fetal and adult brain [77, 81]. Additionally, knockdown of miR-21 in glioblastoma cells leads to
caspase activation and increased apoptosis in vitro and is
able to synergize with neural precursor delivered S-TRAIL
to inhibit glioma cell proliferation and survival in vivo [81].
In contrast to miR-21 overexpression, several miRNAs
have been implicated as tumour suppressors in brain tumors
as a result of their observed downregulation. miR-124 and
miR-137 have been reported to exhibit reduced expression in
anaplastic astrocytomas and glioblastoma multiforme. Their
exogenous expression in either normal neural or brain tumor
stem cells promotes morphological and expression changes
consistent with cellular differentiation [82]. miR-124
expression has also been shown to be reduced in
medulloblastomas and re-expression of miR-124 in a
medulloblastoma cell line resulted in downregulation of the
CDK6 oncogene and slowed proliferation [83]. A very recent
report identified miR-128 as significantly downregulated in
primary glioblastomas compared to adjacent normal tissue
devoid of tumor cells [84]. This study implicated miR-128 as
a putative tumor suppressor in glioblastoma capable of
repressing the oncogenic properties of Bmi1 and inhibiting
self-renewal of glioma stem-like cells [84].
We have recently shown that the miRNA-17/92 polycistron is amplified and upregulated in Sonic Hedgehog-driven
medulloblastomas, and is induced by N-Myc in Sonic
Hedgehog-treated cerebellar neural precursor cells [85].

Back to the future: advanced cancer genome

sequencing
Recent technical breakthroughs in DNA sequencing platforms and their utility in genomic analyses have resulted in

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a number of coordinated efforts focused on comprehensive

characterization of the cancer genome. Indeed The Cancer
Genome Atlas (TCGA), led by the National Cancer Institute
and the National Human Genome Research Institute, is a
multi-institutional consortium aimed at improving our
understanding of cancer through genomic characterization
and high-throughput next-generation sequencing technologies. The first study published by the TCGA involved
integrated DNA copy number, gene expression, and DNA
methylation profiles of 206 glioblastomas [45]. PCRdirected exon re-sequencing was performed for 601 selected
genes in 91 of the tumours. This approach of integrating
multi-dimensional genomic data from glioblastomas
revealed the consistent deregulation of p53, RB, and PI(3)
kinase signaling pathways in the majority of tumors and
established a connection between MGMT promoter methylation and a hypermutator phenotype in some cases [45]. In
a similar study, 22 glioblastomas were comprehensively
profiled by exon re-sequencing of 20,661 protein-coding
genes, analyzed for copy number aberrations using SNP
arrays, and gene expression by combining SAGE (Serial
Analysis of Gene Expression) with next-generation
sequencing [86]. This report identified recurrent mutations
in IDH1 in 12% of GBM patients, mostly in young patients
and those with secondary GBMs, and showed that patients
harboring IDH1 mutations had increased overall survival.
Although both of the studies mentioned above relied
primarily on PCR-based Sanger-sequencing, many groups
are now taking advantage of next-generation/next-gen or
deep sequencing methods for studying normal and disease genomes. There are currently three main next-gen
platforms on the market: 454 (Roche), Solexa (Illumina),
and SOLiD (Applied Biosystems). 454 and Solexa are
based on sequencing by synthesis chemistries and SOLiD
involves sequencing by ligation (Reviewed in [87]).
Despite differences in sample preparation, sequencing
chemistry, and read length, the end result is up to tens of
gigabases of sequencing data from a single run.
The high-throughput afforded by next-gen sequencing
has recently enabled several significant breakthroughs in
genomics. For example, when combined with array-mediated exon capture technology, targeted re-sequencing of
*200,000 protein-coding exons in a single experiment has
been achieved [88]. In addition, using a paired-end strategy
to generate sequence reads from both ends of millions of
DNA fragments, Campbell et al. [89] identified [100
somatically acquired genomic rearrangements in human
lung cancer cell lines. Other studies have exploited next-gen
sequencing to identify transcription factor targets and map
histone modifications using ChIP-Seq [9092] and measure
gene expression profiles and detect alternative splicing
using mRNA-Seq [93, 94]. It can be anticipated that many
of these novel sequencing-based methods will be routinely

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J Neurooncol (2009) 92:261273

applied in cancer genomics, including the study of brain

tumours in the near future.

Children, and B.r.a.i.n child. Paul Kongkham is a recipient of a

National Cancer Institute of Canada Terry Fox Foundation research
fellowship.

Conclusions

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How are these molecular applications influencing patient

diagnosis and treatment today? As some examples, the loss
of 1p and 19q in anaplastic oligodendrogliomas affords
patients a survival advantage while on temozolomide
chemotherapy; methylation of the MGMT promoter is predictive of response to temozolomide and radiation therapy
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FISH in a posterior fossa tumor in a child establishes the
diagnosis of ATRT; and the presence of nuclear beta-catenin
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As new genetic disturbances are uncovered through
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genetic platforms and tools that are available. Hence, we
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