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Report/publication of the evaluation of the
in silico experiments including embryological and neonatalogical
considerations
Project Number: FP7--IST-223979 Deliverable id: D 9.4 Deliverable name: Report/publication of the evaluation of the in silico experiments
including embryological and neonatalogical considerations Submission Date: 30/09/2011
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and neonatalogical considerations
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COVER AND CONTROL PAGE OF DOCUMENT
Project Acronym: CONTRA CANCRUM
Project Full Name: Clinically Oriented Translational Cancer Multilevel Modelling
Document id: D 9.4
Document name: Report/publication of the evaluation of the in silico experiments including embryological and neonatalogical considerations
Document type (PU, INT, RE)
PU
Version: 1.1
Submission date: 30/09/2011
Editor: Organisation: Email:
Norbert Graf USAAR [email protected]
Document type PU = public, INT = internal, RE = restricted
ABSTRACT: ContraCancrum is developing a multilevel platform for simulating malignant tumour development and response to treatment. This deliverable deals with tumour growth and organism development. The comparison between tumor growth and embryonal development of normal tissue is of utmost importance for the understanding of the biocomplexity of cancer. This deliverable solely concentrates on brain tumours.
KEYWORD LIST: Tumor growth, embryological development
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MODIFICATION CONTROL
Version Date Status Author
0.1 15/03/2010 Draft Norbert Graf
0.5 15/06/2011 Draft Eckart Meese
0.8 14/07/2011 Draft Eckart Meese
0.9 17/07/2011 Draft Norbert Graf
1.0 12/09/2011 Final Norbert Graf
1.1 30/9/2011 Final – Update Norbert Graf
List of Contributors
− Norbert Graf, USAAR
− Eckart Meese, USAAR
− Yoo-Jin Kim, USAAR
− Holger Stenzhorn, USAAR
− Georgios Stamatakos, ICCS
− Amos Folarin, UCL
− Christina Backes, USAAR
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Contents 1 EXECUTIVE SUMMARY .............................................................................................................................. 5 2 INTRODUCTION ............................................................................................................................................ 6 3 BRAIN DEVELOPMENT ............................................................................................................................... 7
3.1 EMBRYOLOGY OF THE BRAIN ..................................................................................................................... 7 4 MOLECULAR BIOLOGY OF GLIOMAS ....................................................................................................13
4.1 ANTIBODY RESPONSE AGAINST GLIOMA ANTIGENS .................................................................................. 16 4.2 GENE EXPRESSION PROFILES .................................................................................................................... 17
4.2.1 Genes enrolled in brain development .............................................................................................. 18 4.2.2 Gene expression profiling ................................................................................................................ 19 4.2.3 Analogies between embryological development and tumor development ........................................ 23 4.2.4 References........................................................................................................................................ 26
5 REFERENCES FOR FURTHER READING .................................................................................................31 6 CONCLUSION ...............................................................................................................................................35 7 APPENDICES .................................................................................................................................................37
Appendix 1 - Abbreviations and acronyms ................................................................................................... 37
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1 Executive Summary Cancer modelling has just started compared to other modelling domains as heart and
liver. Though cancer is a very complex and intrinsically variable biological
phenomenon it is expected that cancer modelling will contribute to better and more
individualized treatments for patients. The cancer models span from modelling
molecular pathways to the modelling of progression or regression of whole tumours
in single patients. To understand the biocomplexity of cancer the normal
embryological development of organs and tissues serves as an important source of
knowledge.
This deliverable focuses solely on the embryologic development of the brain and the
impact on the understanding of the brain tumours.
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2 Introduction Human gliomas are the most common primary central nervous system neoplasms. The mechanisms leading to malignant gliomas are poorly understood. Glioma tumour
cell growth (cell divisions and differentiation), vascularisation, and invasiveness (cell
migration) are the most important steps in the development of gliomas. Embryology
might provide insight into neoplastic development in general.
In the developing nervous system there must be:
• ability of neuroblasts to divide, to differentiate and to migrate
• vascularisation and nutritional supply
• ability of axons of neurons to go to an appropriate location
• ability to set up synaptic circuits
• consolidation and maturation of these neuronal connections
The understanding of the molecular mechanisms in brain development, especially
cell division and differentiation, migration, vascularisation and apoptosis will have a
great impact in the understanding of brain tumours. Regarding molecular
pathogenesis the main focus has to be put on cell-cell interactions, cell division and
differentiation, migration and vascularisation. It is important to consider that during
development of the nervous system 50% of the neurons die due to apoptosis.
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3 Brain Development
3.1 Embryology of the brain
The normal development of the brain starts with the thickening of the dorsal
ectodermal surface of the early embryo which will elongate to form the neural plate at
day 16. Subsequent changes convert the plate into a neural tube. Cells lining the
cavity of the neural tube are neuroepithelial cells which ultimately give rise to all the
cells of the CNS. The anterior end of the neural plate enlarges and will form the brain
while the caudal portion of the neural plate will form the spinal cord. The cavity of the
developing neural tube is destined to become the ventricular system. Ectoderm at the
margins of the neural folds represents cells destined to become neural crest cells
that form clusters. These dorsally located cells migrate peripherally to become
sensory ganglia of the cranial and spinal nerves, autonomic and enteric ganglia,
Schwann cells, melanocytes, adrenal chromaffin cells, and the pia/arachnoid
membranes, whereas the dura develops from the mesoderm. By about 25 days the
cephalic and caudal closure of the neural tube becomes complete. The cephalic
closure of the neural tube is marked by the lamina terminalis of the adult. After
closure of the neural tube, the expanded rostral portion of the neural tube will
differentiate and subdivide into three vesicles representing the forebrain
(prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon).
These bends are due to tremendous cell proliferation, differential growth, and
because the brain develops in the confined space of the cranial vault. By about the
end of the 3rd week this 3 part brain begins to assume a “C”-shape by the formation
of cephalic flexure at the level of the mesencephalon; at the end of the 4th week a
cervical flexure develops between the hindbrain and spinal cord. By the end of the
4th week the 3 part brain begins to develop 5 vesicles (Fig. 1). The forebrain
(prosencephalon) gives rise to,
• the paired lateral telencephalic vesicles which bud off from the
prosencephalon and will become the cerebral hemispheres and
• the diencephalon (from which the optic vesicles extend).
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At the cephalic flexure, the mesencephalon remains tubular and undivided. The
hindbrain (rhombencephalon) subdivides into a metencephalon and a more caudal
myelencephalon. In the 5 vesicle stage (6th week) the pontine flexure develops in the
rhombencephalon which divides it into a metencephalon and myelencephalon. The
metencephalon is more cranial and forms the pons and cerebellum; the
myelencephalon becomes the medulla. The presumptive site of the cerebellum is
seen as the rhombic lips at the cranial edge of the thin roof of the 4th ventricle. A
depression develops in the prosencephalon which defines the telencephalon from the
diencephalon. Subsequent growth of the telencephalon will cause it to expand
dorsally, caudally, laterally, and inferiorly.
Fig. 1. Five vesicle stage of brain development.
As these enlargements appear, the brain begins to curve in certain areas (Fig. 2).
There is a cephalic flexure that appears in the region of the midbrain or
mesencephalon. The pontine flexure is located at the junction between the
metencephalon and the mylencephalon. It will eventually give rise to the rhombic lip
of the metencephalic alar plate and then become the cerebellum. There is also a
Telencephalon Cerebral Hemisphere
Diencephalon Diencephalon
Mesencephalon Midbrain
Metencephalon Pons and cerebellum
Myelencephalon Medulla
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cervical flexure that appears at the medulla-spinal cord junction.
The internal central canal of the neural tube in the region of the brain enlarges and
except for the midbrain develops into ventricles. The ventricles will contain the
cerebrospinal fluid. The ventral horn cells of the spinal cord constitute a cell column
that runs the length of the cord, and contains somatic efferent neurons that are
involved in motor innervation to skeletal muscles. Sensory neurons of the spinal cord
form cell columns in the dorsal horn of the spinal cord. Finally, autonomic motor
neurons form a cell column that is located in the lateral gray horn and is located from
T1 to L2.
Fig. 2. Formation of flexures.
The mylencephalon develops into the medulla. The caudal end of the
mylencephalon remains closed with a central canal continuous with the central canal
of the spinal cord. As the more cephalic end of the medulla is reached, the roof of the
ventricle opens and is drawn out as the roof plate. Together with the pia the roof
plate gives rise to the tela choroidea. Heavily vascularised parts of the tela choroidea
project into the 4th ventricle as the choroid plexus.
cerebellum
metencephalon
mylencephalon
spinal cord
telencephalon
diencephalon
mesencephalon
cerebellum
metencephalon
mylencephalon
spinal cord
telencephalon
diencephalon
mesencephalon
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The low medulla contains a central canal and the cell columns that develop are for
the hypoglossal nerve (CN12), the vagus nerve (CN10) and the sensory nuclei for
the sensory nuclei for cranial nerves 10 and 5 (the trigeminal nerve). New pathways
that ascend and descend through the spinal cord are located posterior and lateral in
the low medulla.
The high medulla sees some changes. As mentioned above the fourth ventricle
opens. Cell columns for the vestibular nuclei and the cochlear nuclei appear. The
sensory nuclei for cranial nerve nuclei 5, 9 (glossopharyngeal nerve) and 10 also are
found. Motor nuclei for the glossopharyngeal and vagus nerves as well as the 12th
cranial nerve are also present. The long ascending and descending tracts have
assumed a more anterior and lateral position.
The metencephalon develops into the cerebellum and the pons. The cephalic part of
the fourth ventricle continues into the pons. The roof plate becomes the superior
medullary velum a region of the pons that will be covered by the cerebellum. During
the formation of the cerebellum the mantle layer of the rhombic lip will develop into
the deep nuclei of the cerebellum. It will also give rise to the cells that will form the
cerebellar cortex.
The cell columns of the pons include those for the sensory nuclei for the
vestibulocochlear nerve, the trigemminal nerve, the facial nerve and the motor nuclei
for the facial nerve, the trigemminal nerve and the abducens nerve. Descending
pathways are located in the base of the pons along with pontine nuclei. Other
ascending pathways are located in the anterolateral pons, and the middle and
superior cerebellar peduncles.
The mesencephalon changes very little during development. The central canal is
small and is now called the cerebral aquaduct. The roof plate and the alar plate
becomes the tectum and contain nuclei associated with vision and with hearing. The
floor and the basal plates become the cerebral peduncles.
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The cell columns associated with the midbrain include those associated with the eye
muscles as well as with autonomic function. The newer ascending and descending
pathways are found along the anterolateral tegmentum and crus cerebri portions of
the midbrain. This part of the brain develops into the thalamus, the epithalamus, the
subthalamus and the hypothalamus. The central canal forms the third ventricle. The
roof plate forms the tela choroidea and choroid plexus and is continuous with the
lateral ventricle via the interventricular foramina of Monroe.
The diencephalon does not have a basal plate. The alar plate divides into a dorsal
region and a ventral region and the dividing line is the hypothalamic sulcus. The
thalamus is located in the dorsal region and constitutes a major set of relay nuclei for
pathways involved in sensory function. The ventral region is comprised of
hypothalamic nuclei. These nuclei are involved in the regulation of visceral function.
During development the telencephalon expands significantly. During their
development they flex into a "C-shaped" structure. The mantle layer gives rise to the
cells of the basal ganglia a region of the brain that regulates motor function. The
mantle layer also gives rise to cells that will migrate into the marginal layer and these
cells become the cortical gray cells. During development the medial walls of the
telencephalon fuse with the lateral walls of the diencephalon. The internal capsule
forms at the line of fusion. The growth of the telencephalic hemispheres causes them
to grow over the basal ganglia and the cortex over it called the insular cortex. The
central canal of the telencephalon forms the lateral ventricle. The choroid plexus
develops along the medial wall of the lateral ventricle. A corpus callosum forms as a
group of fibers that connect one hemisphere to the other.
During histogenesis ectodermal cells of the early tube develop 3 concentric zones:
• germinal (or matrix),
• mantle, and
• marginal
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Cells of the original single-layered tube divide to form a neuroepithelium whose cells
extend from the neural canal to the tube’s external surface. Cells near the central
canal are called the germinal layer. They divide rapidly, thickening the walls of the
tube and eventually producing neuroblasts and glioblasts. Newly formed
undifferentiated cells of the neuroepithelium migrate outward forming a 3-layered
tube consisting of
• an internal, columnar ependymal layer
o becomes the ependymal lining and epithelium of choroid plexus,
• a middle, densely packed layer of mantle cells
o becomes the gray matter of the CNS, and
• an external marginal layer composed mainly of the processes of cells of the
mantle layers
o becomes the white matter of the CNS
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4 Molecular biology of gliomas
Morphogens seem to play unexpected roles in the development and degeneration of
the central nervous system. Homeobox genes as well as signaling factors play a
major role in this setting. In early development, embryos transform from a simple
group of cells into an organism of complex shape. This occurs through a precise re-
arrangement and re-positioning of cells and tissues. Therefore the tracking of how
this process of shaping occurs is expected to help understanding what goes wrong in
cancer. Many of the steps used in normal shaping of embryos reappear in an
uncontrolled fashion in malignant tumours. While surgery, radiation therapy, and
chemotherapy have roles to play in the treatment of patients with gliomas; these
therapies are self-limited because of the intrinsic resistance of glioma cells to therapy
and the diffusely infiltrating nature of the lesions. It is now known that malignant
gliomas arise from a number of well-characterized genetic alterations, activations of
oncogenes and inactivation of tumour suppressor genes.
The recognition of the putative brain tumour stem cell, the tumour initiating cell in
brain cancer, provides posiible targets for new drugs and a more efficient and less
toxic treatment. Τable 1 shows proto-oncogenes involved in gliomas. Table 2 shows
several common tumour suppressor genes involved in gliomas. Table 3 presents
some common genetic aberrations in familial astrocytic tumours. Bansal et al.1
described the glioma signaling pathway in a review on the molecular biology of
human gliomas. It has long been accepted that tumours are dependant on
angiogenesis, the directional sprouting of new blood vessels from existing vessels
within the tumour2
1 Bansal K, Liang ML, Rutka JT: Molecular biology of Human gliomas. Technology in Cancer Res Treatment 5: 185-194, 2006
. During embryogenesis, the normal physiologic creation of new
vasculature is termed vasculogenesis, whereas the budding of capillaries from
2 Ekstrand AJ, James CD, Cavenee WK, et al.: Genes for Epidermal Growth-Factor Receptor, Transforming Growth Factor-Alpha, and Epidermal Growth-Factor and Their Expression in Human Gliomas In Vivo. Cancer Res 51: 2164-2172, 1991
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existing blood vessels is called angiogenesis3. Tumours must secrete diffusible
chemicals that stimulate endothelial cells via a paracrine effect and leads principal
cell type contributes to angiogenesis. The process of angiogenesis has been
quantified by measuring microvessel density in tumour specimens4
.
Table 1. Proto-oncogenes involved in gliomas5
Gene
Location Typical alteration Function of the protein Common in
EGFR 7p11 Amplification and overexpression
genomic rearrangement
Tyrosin kinase growth factor receptor
Glioblastoma (GBM)
PDGFR 4q12 Amplification and overexpression
Tyrosin kinase growth factor receptor
GBM
MET 7q31 Amplification and overexpression
Tyrosin kinase growth factor receptor
GBM
CDK4 12q13 Amplification and overexpression
Cyclin-dependent kinase, promotes G1/S phase progression
GBM
CCND1 11q13 Amplification and overexpression
Cyclin D1, promotes G1/S phase progression
GBM
CCND3 6p21 Amplification and overexpression
Cyclin D3, promotes G1/S phase progression
GBM
MDM2 12q15 Amplification and overexpression
Inhibitor of p53 function GBM
MDM4 1q32 Amplification and overexpression
Inhibitor of p53 function GBM
MYCC 8q24 Amplification and overexpression
Transcription factor GBM
3 Guha A, Dashner K, Black PM, et al.: Expression of Pdgf and Pdgf Receptors in Human Astrocytoma Operation Specimens Supports the Existence of an Autocrine Loop. International Journal of Cancer 6:168-173, 1995 4 Hermanson M, Funa K, Hartman M, et al.: Platelet-Derived Growth-Factor and Its Receptors in Human Glioma Tissue – Expression of Messenger-RNA and Protein Suggests the Presence of Autocrine and Paracrine Loops. Cancer Res 52: 3213-3219, 1992 5 Bansal K, Liang ML, Rutka JT: Molecular biology of Human gliomas. Technology in Cancer Res Treatment 5: 185-194, 2006
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Table 2. Common tumour suppressor genes involved in gliomas5
Gene Location Typical alteration
Protein Function Commonly Seen In
TP53 17p13 Mutation Involved in the regulation of apoptosis, cell cycle progression, DNA repair
Anaplastic astrocytomas, glioblastoma (secondary > primary)
PTEN 10q23 Mutation Protein phosphatase and lipid phosphatase, negative regulator of phosphatidylinositol 3-kinase
Glioblastoma
CDKN2A 9p21 Homozygous deletion
Inhibitor of cyclin dependent kinase 4 and 6
Glioblastoma
RB1 13q14 Mutation hypermethylation
Nuclear phosphoprotein involved in cell cycle regulation
Glioblastoma, Anaplastic astrocytomas
p14 9q21 ARF Homozygous deletion hypermethylation
Inhibitor of Mdm2 Anaplastic astrocytomas
Table 3. Common genetic aberrations in familial astrocytic tumours
5
Syndrome
Gene Location Function Tumour type
NF-1 NF1 17q11.2 GTPase activating protein homology
Astrocytic tumors (brain stem optic n.) ependymomas
NF-2 NF2 22q12.2 Ezrin/Moesin/Radixin-like
Vestibular Schwannomas, gliomas
Li Fraumeni TP53 17p13.1 Transcription factor Apoptosis inducer
Astrocytic tumors
Tuberous sclerosis
TSC1/2 9q34/16p13.3 GTPase activating protein homology
Subependymal Giant cell astrocytoma
Turcot’s syndrome
MLH1/PS2 3p21.3/7p22 MIN+ Glioblastoma
Cowden disease PTEN 10q22-q23 Dual-specificity phosphatase and Tensin homology
Astrocytic tumors
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4.1 Antibody response against glioma antigens
Recently there is increasing evidence for antibody response against proteins
expressed in human tumours. Tumour cells are characterized by altered pathways
and/or altered proteins that can be recognized by the human immune system.
Antibodies that can be detected in patients’ blood offer a unique possibility to monitor
tumour development without being dependent on tumour biopsies. Most recently,
multiple antibodies were found for several human tumours indicating a complex
humoral immune response in tumour patients. In 2005 complex seroreactivity
patterns were reported for human tumours including one study on prostate cancer
and the other study by our own group (USAAR) on meningioma which is a generally
benign human cranial tumour. We not only reported first evidence for specific
seroreactivity pattern in benign tumours but also evidence for reactivity pattern
specific for tumour subtypes6
.
6 Comtesse N, Zippel A, Walle S, et al.: Complex humoral immune response against a benign tumor: frequent antibody response against specific antigens as diagnostic targets. Proc Natl Acad Sci USA 102:9601-9606, 2005
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4.2 Gene expression profiles
New analysis technologies, e.g., DNA microarrays, have improved the knowledge of
cancer development. More informative technologies like gene expression profiling
clearly show the molecular heterogeneity of tumors although their morphological
characteristics are exactly defined. In general, gliomas, e.g., can be sub-divided into
two major groups: primary glioma arising de novo and secondary glioma developing
from low grade glioma [1]. This new molecular methods allow classifying tumors in
another way. Consequently, gliomas can be divided into different molecular
subtypes, e.g., the classification of glioblastoma into proneural, neural, classical, and
mesenchymal subtypes [2].
Oncogenes like CDK4, EGFR, and MDM2 are known to be overexpressed in gliomas
whereas tumor suppressor genes like CDKN2A, PTEN, RB1, and TP53 are weakly
expressed in this tumor tissue. For the last decades, several studies focused on gene
expression profile could find further genes with abnormal expression profiles in
gliomas. Gene expression profiles disclose differences in gene expression between
normal brain tissue and tumor tissue as well as between oligodendroglial tumors and
Glioblastoma multiforme. A study published in 2007 identified 187 genes with
different expressions in different types of gliomas: Genes like SOX4 (Genbank
accession no. NM_003107), BRSK2 (Genbank accession no. NM_003957), and
KIAA0471 (Genbank accession no. AB007940) show higher expression in anaplastic
oligodendrogliomas. Genes like MYL6 (Genbank accession no. NM_079423), CD14
(Genbank accession no. NM_000591), and GPI (Genbank accession no.
NM_000175) were higher expressed in glioblastomas [3].
Interestingly, gene expression patterns of samples from different region of the same
tumor bear a resemblance although the tumor regions clearly differ in
histopathological characteristics [4]. The expression pattern of certain gens seems to
correspondent with the survival time: A high expression leads to a rapid tumor
development [4]. Moreover, certain genes playing a role in tumor development also
show high activity during the embryological development of the central nervous
system [4].
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4.2.1 Genes enrolled in brain development
During embryogenesis the brain develops from neuroepithelial cells. Neural stem
cells play an important part in this development. These pluripotent cells differentiate
to both glial cell and neural cell types. During early embryogenesis these neural stem
cells can be identified by the expression pattern of the protein nestin [5-7].
Furthermore, a set of several hundred genes is involved in this complex development
process as gene expression profiles could reveal in the last years. The genes are
responsible for products like signaling, housekeeping, metabolic, cytoskeletal, and
neuron specific proteins. Interestingly, most of these genes are only expressed at a
particular time of the development of the central nervous system [8-10]. E.g., during
the formation of the neural tube Gbx2 and Sox1 have been observed to be
expressed in an unregulated way whereas Gbx2 is down-regulated when the neural
tube is finished [11,12]. Genes like Sox2 and N-Cam are found over-expressed in
undifferentiated neurectoderm and neural progenitors [12,13]. Pax 6 and Shh are up-
regulated, Pax 7 is down-regulated during the enrichment of radial glia cells [14,15].
Further sets of genes, including the family of the Hox genes, are also involved in this
hierarchical regulation of brain development. Furthermore, epigenetic changes as a
necessary part of a correct development can be observed during this process [16].
Neural stem cells which are important for this process were recently detected in sub-
ventricular region of the adult mammalian brain [17]. As during embryogenesis these
cells are able to renew, to proliferate, to migrate, and to generate differentiated
progeny cells. These neural stem cells are supposed to be associated with glioma
development. Especially, stem cells expressing the marker CD133 were reported to
be involved into tumor growth. In culture, these CD133+
cells were able to
differentiate into tumor cells. Most notably, the researchers reported that these tumor
cells bear high analogy to the tumor from the patient [18].
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4.2.2 Gene expression profiling
Gene expression profiling by different high-throughput technologies as microarrays
and next-generation sequencing has become an invaluable method for gaining
insights in the differences of gene expression in disease and control groups. In
recent years, the public available data as well as bioinformatic tools for evaluating
such data increased exponentially. One of the most popular repositories for
expression data in general is the Gene Expression Omnibus (GEO) from the National
Center for Biotechnology Information (NCBI) [19]. GEO was established more than
10 years ago and stores now over 20 000 microarray- and sequence-based
functional genomics studies for a variety of model organisms including for example
human and mouse [20].
Another public resource for high-throughput cancer data is The Cancer Genome
Atlas (TCGA) (http://cancergenome.nih.gov/). The TCGA project is a comprehensive
and coordinated effort to accelerate the understanding of the molecular basis of
cancer through the application of genome analysis technologies, including large-
scale genome sequencing. TCGA is a joint effort of the National Cancer Institute
(NCI) and the National Human Genome Research Institute (NHGRI), two of the 27
Institutes and Centers of the National Institutes of Health, U.S. Department of Health
and Human Services [21].
An exemplary study of Verhaak et al. [2] made use of the TCGA data and
characterized clinically relevant subtypes (Proneural, Neural, Classical and
Mesenchymal) of glioblastoma multiforme (GBM). They extracted an 840 gene
signature (210 per subtype) and showed that based on this gene list different GBM
data sets could be clustered into four relevant subtypes. Taking advantage of the well
defined expression characteristics of the Mesenchymal and Classical phenotypes at
the transcriptional level we implemented a classifier in R based on the four main
subtypes defined in by Verhaak et al in their core sample set. Predictions of subtype
were performed on the ContraCancrum affymetrix (hgu133plus2) datasets. 30 of 40
samples are classified and the prediction data made available to the project
workpackages. The results of the analysis are summarized in Table 4. An example
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classification is shown in Figure 4, as it will be visualized on the ContraCancrum site.
As can be observed, most of our samples are classified in the Classical type (9/30),
followed by Proneural (8/30), Mesenchymal (7/30), and Neural (6/30) (Figure 3).
Classical Proneural Mesenchymal Neural0
5
10
15
20
25
30
%
Fig. 3: Distribution of the PCA classification results for the 30 ContraCancrum affymetrix samples
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Sample Label Classification Group
G4 Proneural
G6 Classical
G8 Proneural
G11 Classical
G13 Proneural
G14 Mesenchymal
G16 Classical
G18 Neural
G50 Mesenchymal
G51 Proneural
G24 Mesenchymal
G25 Classical
G53 Classical
G54 Proneural
G58 Classical
G47 Proneural
G48 Mesenchymal
G36 Classical
G37 Neural
G39 Neural
G42 Classical
G44 Mesenchymal
G45 Proneural
G29 Proneural
G27 Classical
G32 Neural
G23 Neural
G56 Mesenchymal
G70 Neural
G71 Mesenchymal
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Table 4: Results of the PCA classification for the 30 ContraCancrum affymetrix samples
Interestingly, Verhaak et al. also found neural precursor and stem cell marker NES,
as well as Notch (NOTCH3, JAG1, LFNG) and Sonic hedgehog (SMO, GAS1, GLI2)
signaling pathways highly expressed in the Classical subtype [2]. In addition, they
found high expression of oligodendrocytic development genes such as PDGFRA,
NKX2-2 and OLIG2 [22] in the Proneural group, as well as several proneural
development genes such as SOX genes, DCX, DLL3, ASCL1, and TCF4 [23].
Furthermore, a Gene ontology (GO) analysis revealed an involvement in
developmental processes and a previously-identified cell cycle/proliferation signature
[24] for the Proneural subtype. Taken together these findings indicate that there exist
Fig. 4: Principal Component Analysis based on the Verhaak et al. core sample set for the ContraCancrum sample G44. This sample is classified as Messenchymal subtype. The color schemes are Classical=Blue, Messenchymal=Red, Neural=Green and ProNeural=Purple.
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common processes at the intersection between embryonal development and
cancerogenesis.
4.2.3 Analogies between embryological development and tumor development
Several studies could reveal analogies between embryological development and
tumor development. Tumor development is characterized by uncontrolled cell
proliferation and tumor growth whereas embryological development can be seen as a
process with controlled cell proliferation. Especially, embryological stem cells play the
major part during this process. Recently, stem cells were also found in adult tissues,
e.g., in pancreas, liver, bone marrow, and brain [17,25-27]. They are supposed to
exist in all tissues. Hence, it is conjecturable that cancer stem cells derive from
normal stem cells which underwent a transformation [28]. According to the cancer
stem cell theory which already arose in the middle of the last century a small
population of cancer cells (cancer stem cells, CSC) with the abilities of self-renewal
and differentiation – equal to embryonic stem cells – are involved in tumorgenesis.
There is still some concern about this theory and more investigations have to be
done to confirm this hypothesis. CSC were recently detected in several cancer
tissues, e.g., in leukemia, lung cancer, melanomas, and brain tumors [29-33].
Latest studies show several signaling pathways are implicated in both tumor
development and embryological development: the NOTCH signaling pathway, the
Wnt pathway, the TGFß pathway, the Jak/STAT pathway, the MAP-Kinase/ERK
pathway, the PI3K/AKt pathway, and the NFkB pathway [34-45]. Most of these
pathways are involved in proliferation, differentiation, or homeostasis processes in
normal tissue. Mutations in these pathways can lead to uncontrolled proliferation and
differentiation promoting tumorgenesis.
Furthermore, the family of the homeobox genes including, e.g., HOX genes, PAX
genes, SOX genes, and CDX genes, plays a role in development and cancer. During
embryogenesis HOX genes are a necessary part of the morphogenesis and further
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homeobox genes (e.g., PAX, MSX, and CDX) are important for the organogenesis
and the development of several tissues [46-47]. Transcription factors like SOX2 and
OCT4 serve as regulators in embryonic stem cells. As several studies could reveal
homeobox genes seem to be associated with metabolic alterations and different
diseases [48]. Abnormal expression patterns of homeobox genes were found in
several cancer tissues: HSIX1 and SOX2 are overexpressed in breast cancer;
HOXC9 is misexpressed in colon cancer; HOXB7 shows high activity in melanomas
whereas no activity can be detected in melanocytes [49-51]. Homeobox genes are
involved in proliferation and differentiation processes during embryogenesis. In turn,
these processes are also an important part of tumor development. Thus,
deregulations in homeobox genes may lead to cancer development.
Latest studies attract notice to a further set of regulatory molecules which may
control the homeobox genes: the miR-10 family. The miR-10 family belongs to the
microRNAs which are a group of small, non-coding RNAS. They play a role in
posttranscriptional gene regulation and were described for the first time in the in the
1990ies. Genes of miR-10 family are located in the HOX cluster and seems to control
the transcription of HOX genes in mammals [52]. Consequently, miR-10 family is
supposed to take part into development processes. Most notably, deregulated
expression patterns of the miR-10 family were shown in several cancer tissues like in
melanoma, pancreatic cancer, and glioblastoma [53-55]. Currently, it is not clear if
the miR-10 family is responsible for tumor progression, or even for tumor initiation.
Hence, future investigations have to analyze the detailed role of this miRNA family.
Besides signaling pathways and homeobox genes playing a necessary part in
development and cancer epigenetic mechanisms can also be detected in both in
tumorgenesis and embryogenesis. Methylation and demethylation, respectively, are
required processes in normal development and play an important role in genomic
imprinting [56]. Methylation patterns regulate gene expression by inhibiting gene
expression and suppressing transcription. Disruptions of these processes can lead to
abnormalities in embryogenesis [57]. Tissue-specific methylation patterns can be
found in normal mature tissue whereas abnormalities in DNA methylation can be
detected in malignance tissues [58]. DNA hypermethylation of tumor suppressor
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genes and hypomethylation of oncogenes seems to be involved in cancer
development, progression, and metastasis [59-61]. Abnormal methylation pattern
were found in several cancer tissues, e.g., in head and neck squamous cell
carcinomas, gastric carcinoma, colorectal cancer, and glioma [60,62-65]. Epigenetic
mechanisms like DNA methylation are essential factors to maintain the genomic
stability. Alteration in this mechanism may advance tumor development.
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G27. Heckel D, Brass N, Fischer U et al.: cDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patients. Hum. Mol. Genet. 6:2031–2041, 1997 G28. Brass N, Ukena I, Remberger K et al.: DNA amplification on chromosome 3q26.1-q26.3 in squamous cell carcinoma of the lung detected by reverse chromosome painting. Eur J Cancer 32A:1205-1208, 1996 G29. Fischer U, Struss AK, Hemmer D, et al.: Glioma-expressed antigen 2 (GLEA2): a novel protein that can elicit immune responses in glioblastoma patients and some controls. Clin Exp Immunol 126:206-213 2001, G30. Comtesse N, Niedermayer I, Glass B et al.: MGEA6 is tumor-specific overexpressed and frequently recognized by patient-serum antibodies. Oncogene 21:239-247, 2002 G31. Heckel D, Brass N, Fischer U, et al.: cDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patients. Hum Mol Genet 6:2031-2041, 1997 G32. Heckel D, Comtesse N, Brass N, et al.: Novel immunogenic antigen homologous to hyaluronidase in meningioma. Hum Mol Genet 7:1859-1872, 1998 G33. Brass N, Racz A, Bauer C, et al.: Role of amplified genes in the production of autoantibodies. Blood 93:2158-2166, 1999 G34. Comtesse N, Heckel D, Racz A et al.: Five novel immunogenic antigens in meningioma: cloning, expression analysis, and chromosomal mapping. Clin Cancer Res 5:3560-3568, 1999 G35. Bauer C, Diesinger I, Brass N, et al.: Translation initiation factor eIF-4G is immunogenic, overexpressed, and amplified in patients with squamous cell lung carcinoma. Cancer 92:822-829, 2001 G36. Monz D, Munnia A, Comtesse N, et al.: Novel tankyrase-related gene detected with meningioma-specific sera. Clin Cancer Res 7:113-119, 2001 G37. Struss AK, Romeike BF, Munnia A, et al.: PHF3-specific antibody responses in over 60% of patients with glioblastoma multiforme. Oncogene 20:4107-4114, 2001 G38. Diesinger I, Bauer C, Brass N, et al.: Toward a more complete recognition of immunoreactive antigens in squamous cell lung carcinoma. Int J Cancer 102:372-378, 2002 G39. Pallasch CP, Struss AK, Munia A, et al.: Autoantibodies against GLEA2 and PHF3 in glioblastoma: tumor-associated autoantibodies correlated with prolonged survival. Int J Cancer 117:456-459, 2005 G40. Dönnes P, Hoglund A, Sturm M, et al.: ntegrative analysis of cancer-related
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data using CAP. FASEB J18:1465-1467, 2004 G41. Backes C, Kuentzer J, Lenhof HP, et al.: GraBCas: a bioinformatics tool for score-based prediction of Caspase- and Granzyme B-cleavage sites in protein sequences. Nucleic Acids Res 33(Web Server issue):W208-213, 2005 G42. Wang X, Yu J, Sreekumar A, et al.: Autoantibody signatures in prostate cancer. N Engl J Med 353:1224-1235, 2005 G43. Kleihues P, Sobin LH: World Health Organization classification of tumors. Cancer 88:2887, 2000 G44. Kleihues P, Ohgaki H: Phenotype vs genotype in the evolution of astrocytic brain tumors. Toxicol Pathol 28:164-170, 2000 G45. Graf N, Hoppe A: What are the expectations of a Clinician from In Silico Oncology ? Edts.: Kostas M, Stamatakos G: Proceedings: 2nd International Advanced Research Workshop on In Silico Oncology (IARWISO), Kolympari, Chania, Greece , 25th and 26th September 2006 G46. For more information regarding embryology of the brain see: http://isc.temple.edu/neuroanatomy/lab/embryo_new/ G47. A growth simulation of Human Embryo Brain is described by S. Czanner, R.Durikovic and H. Inoue (Software Department, The University of Aizu; Aizu-Wakamatsu City, Japan) and can be found at: http://cgg-journal.com/2001-3/02/SCCG2001.htm http://doi.ieeecomputersociety.org/10.1109/SCCG.2001.945348
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6 Conclusion In a paper published by Dahmane et al. in Development and Disease in 2001 about
the Sonic Hedgehog-Gli pathway7
In summary we found that it seems to be obvious that embryological development
and cancer development have something in common. As a consequence it is also
comprehensible that there seems to be an analogy between glioma development and
brain development. Recent studies reveal that several genes seem to be involved in
the embryological brain development as well as in the tumor growth. In this study, we
are not able to show correspondence between glioma and brain development. In
future, further and more detailed investigations have to be done to obtain extensive
knowledge about these molecular processes. Especially, the biology of neural stem
they conclude: „The mechanisms that regulate the
growth of the brain remain unclear. We show that Sonic hedgehog (Shh) is
expressed in a layer-specific manner in the perinatal mouse neocortex and tectum,
whereas the Gli genes, which are targets and mediators of SHH signaling, are
expressed in proliferative zones. In vitro and in vivo assays show that SHH is a
mitogen for neocortical and tectal precursors and that it modulates cell proliferation in
the dorsal brain. Together with its role in the cerebellum, our findings indicate that
SHH signaling unexpectedly controls the development of the three major dorsal brain
structures. We also show that a variety of primary human brain tumors and tumor
lines consistently express the GLI genes and that cyclopamine, a SHH signaling
inhibitor, inhibits the proliferation of tumor cells. Using the in vivo tadpole assay
system, we further show that misexpression of GLI1 induces CNS hyperproliferation
that depends on the activation of endogenous Gli1 function. SHH-GLI signaling thus
modulates normal dorsal brain growth by controlling precursor proliferation, an
evolutionarily important and plastic process that is deregulated in brain tumors.” This
conclusion started a discussion about the enrolment of genes in brain development
that are also involved in the development of gliomas. The thesis is that understanding
brain development from molecular biology will help to understand the molecular
biology of gliomas and will find new targets for drug development for treatment of
gliomas.
7 Dahmane N et al.: The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development 128: 5201-5212, 2001
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cells and their potential of migration and motility may pave the way to understand the
biological processes during cancerogenesis. The findings will allow new opportunities
to get a detailed look into tumorgenesis. Moreover, they provide a new way for future
therapy targets.
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7 Appendices Appendix 1 - Abbreviations and acronyms
CSC Cancer Stem Cell
CSF Cerebrospinal fluid
EGFR Epidermal Growth Factor Receptor
GBM Glioblastoma multiforme
GEO Gene Expression Omnibus
GM Gray matter
GO Gene ontology
NCBI National Center for Biotechnology Information
NCI National Cancer Institute
NHGRI National Human Genome Research Institute
TCGA The Cancer Genome Atlas
WM White matter