Report/publication of the evaluation of the in silico ... · in silico experiments including embryological and neonatalogical considerations. ... Deliverable name: Report/publication

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

CONTRA CANCRUM FP7-223979 D9.4 – Report/publication of the evaluation of the in silico experiments including embryological

and neonatalogical considerations

05/10/2011 Page 2 of 37

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



05/10/2011 Page 14 of 37

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


GBM

MET 7q31 Amplification and overexpression


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].



05/10/2011 Page 18 of 37

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].



05/10/2011 Page 19 of 37

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

http://cancergenome.nih.gov/�



05/10/2011 Page 20 of 37

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



05/10/2011 Page 21 of 37

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.



05/10/2011 Page 23 of 37

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



05/10/2011 Page 25 of 37

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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G13. Demuth T, Berens ME: Molecular Mechanisms of Glioma Cell Migration and Invasion. Neuro-Oncology 70:217-228, 2004 G14. Rooprai HK, Vanmeter T, Panou C, et al.: The Role of Integrin Receptors in Aspects of Glioma Invasion In Vitro. Int J Dev Neurosci 17: 613-623, 1999 G15. Nollet F, Kools P, van Roy F: Phylogenetic Analysis of the Cadherin Superfamily Allows Identification of Six Major Subfamilies Besides Several Solitary Members. J Mol Biol 299: 551-572, 2000 G16. Boda-Heggemann: Zell- und molekularbiologische Charakterisierung neuartiger Zell-Zell-Verbindungsarten in Glioma-Zellen und mesenchymalen Stammzellen. Inaugural-Dissertation, Heidelberg 2005; http://archiv.ub.uni-heidelberg.de/volltextserver/volltexte/2006/6127/pdf/Boda_Heggemann_Dissertation.pdf G17. Santarius T, Kirsch M, Rossi ML, et al.: Molecular Aspects of Neuro-oncology. Clin. Neurol. Neurosurg. 99:184-195, 1997 G18. Folkman J: Angiogenesis in Cancer, Vascular, Rheumatoid and Other Disease. Nat. Med. 1: 27-31:1995 G19. Folkman J: Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical Applications of Research on Angiogenesis. N Eng. J Med 333:1757-1763, 1995 G20. Hara A, Okayasu I: Cyclooxygenase-2 and Inducible Nitric Oxide Synthatase Expression in Human Astrocytic Gliomas: Correlation with Angiogenesis and Prognostic Significance. Acta Neuropathol (Berl) 108:43-48, 2004 G21. Zadeh G, Qian B, Okhowat A, et al:.Targeting the Tie2/Tek Receptor in Astrocytomas. Am. J. Pathol. 164:467-476, 2004 G22. Weinder N: Angiogenesis in Breast Cancer. Cancer Treatment Res 83:265-301, 1996 G23. Li VW, Folkerth RD, Black PM: Microvessel Count and CSF Basic Fibroblast Growth Factor in Children with Brain Tumors. Lancet 344: 82-86, 1994 G24. Leon SP, Folkerth RD, Black PM: Microvessel Density is a Prognostic Indicator for Patients with Astroglial Brain Tumors. Cancer 77: 362-372, 1996 G25. Lassman AB: Molecular Biology of gliomas. Curr Neurol Neurosci Rep 4:228-233, 2004 G26. 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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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

Documents

Report/publication of the evaluation of the in silico ... · in silico experiments including embryological and neonatalogical considerations. ... Deliverable name: Report/publication