CANCER METASTASIS:
A SYSTEMS BIOLOGY APPROACH
BY: CRAIG TSCHIRHART, B.Sc. (ENG)
DATE: 28/10/2003
FOR: DR. DAVIES
COURSE: BME 1450
ASTRACT - Cancer is the second leading cause of death in the
U.S. Breast cancer and prostate cancer are the most common
forms of cancer. Systems biology has been implemented to
better understand the onset, diagnosis, and metastasis of
cancer. The systems biology approach has become one of the
preferred methods of studying the pathology of cancer. A
more comprehensive understanding of the genetic mechanism
of prostate cancer development will be facilitated by further
development of cell-type-specific cDNA libraries.
The
purpose of this study is to investigate the onset of cancer and
the diagnosis and treatment of tumour growth and metastasis
by means of systems biology technology. Research in this
area has made improved methods of distinction between
aggressive and indolent tumours and the role of factors such
as endostatin, EGRF, TM, and other growth factors have
become more clearly understood. The results of this study
indicate that metastasis is dependent on endostatin,
Epidermal
Growth
Factor
Receptor
(EGRF),
Tetrathiomolybdate, and various growth factors.
1.0 INTRODUCTION
One in every four deaths in America is due to cancer, ranking
it second only to heart disease as the leading cause of death in
the U.S [1]. In children between the ages of 1 and 14, cancer
is the leading cause of death. World-wide, there are over 1.33
million cancer-related deaths per year, totally a cost of over
$170 billion [1]. Breast cancer and prostate cancer are the
most common forms of cancer detected for males and females
respectively, followed by lung/bronchus, and colon cancer for
both males and females. Statistically, the most harmful type
is lung/bronchus cancer, which accounts for approximately
28% of all cancer deaths [1].
Systems biology refers to the study of biological systems to
formulate mathematical models to describe the structure and
behaviour of the system [2]. Rather than analysis of
individual genes or proteins, systems biology considers the
function of all of the elements in the system. This entails
biological, genetic, or chemical perturbation of the system;
monitoring of the gene, protein, and informational pathway
responses; and analytic observation of the data [2]. The data
can then be integrated, graphically displayed, and finally
modelled computationally [2].
Nearly all cancer cells are generated from a series of genetic
DNA mutations accumulating within an otherwise healthy cell
[3]. These mutations may be hereditary or they may arise
from external sources, such as smoking, environmental toxins,
or viruses. Mutations may also occur from mistakes in DNA
repair with increased age [3]. The nanotechnology associated
with systems biology facilitates a more precise diagnosis,
specific to the gene in which it is expressed. This increases
the specificity of diagnosis so that more specific treatment
options may be made available. Currently, chemotherapy and
radiation are the leading treatment options. However, these
are quite generic options [3]. As research in this area
continues, more effective gene-specific drugs are anticipated
to become the new leading treatment method. Further,
molecular-based diagnosis will allow for cancer detection
using a small cell sample at a low cost very quickly [3].
A plethora of studies are being conducted to improve
understanding, curing, prevention, and spread of cancer.
Metastasis refers to the migration of tumour tissue to one or
more sites throughout the body. The systems biology
approach will allow for accurate identification of different
types and stages of cancer early in the transformation process,
and to understand the interplay between genetic and
environmental networks exposed to cancer. Ideally, these
studies will facilitate personalized treatment and early
diagnosis of people at risk. The purpose of this study is to
investigate the onset of cancer and the diagnosis and treatment
of tumour growth and metastasis by means of systems biology
technology.
2.0 BACKGROUND
2.1 Onset of Cancer
Cancer cells are caused by a mutation or irreparable damage
to DNA [1]. This damage may be inherited or due to
environmental factors. Unlike a normal cell, which tends to
divide only when it is dying or damaged, a cancer cell tends to
multiply and continue to form additional abnormal cells
without necessarily being damaged. The rapid reproduction
of cancer cells eventually leads to the onset of a tumour or
migration through the bloodstream or lymph vessels [1].
Migration of malignant tumour tissue is referred to as
metastasis.
2.2 Goals of Systems Biology
The first goal of systems biology research is to identify all of
the elements within a system and to create a database
containing that information [2]. This approach is referred to
as discovery science. When combined with hypothesis-driven
science, the means of gene expression becomes more clearly
understood. The information relating to gene expression is
hierarchical in nature (Figure 1). Once information is
collected at each of these levels for individual biological
systems, the data may be integrated to generate analytical
mathematical models of the system(s) [2].
mRNA
DNA
Informational
Pathways
Tissues
Protein
Protein
Interactions
Informational
Networks
Organism
Cells
Ecosystems
Figure 1: Hierarchical Levels of Biological
Information [2]
Recent developments in software and global experimental
techniques have allowed for identification of gene locations
within in a sequenced genome. As the genomic sequence is
revealed, the adjacent regulatory sequences become
accessible. In turn, a comprehensive understanding of
polymorphisms may be developed, some of which are
responsible for differences in physiology and disease
predisposition [2].
3.0 RESULTS AND DISCUSSION
3.1 Tumour Distinction: Aggressive Versus Indolent
Cancerous Tissue
One of the difficulties associated with tumours once they have
been detected is being able to distinguish between aggressive
or malignant tumours and indolent or benign tumours [3]. It
has become apparent that initial prostate carcinomas growth is
dependent on androgen. Therefore androgen ablation therapy
has been successful in treating indolent tissue. However,
malignant tumours tend to lose their dependence on androgen
eventually, and begin androgen-independent growth [3].
The mechanism for progression from androgen dependence to
independence is not well understood. However, studies are
being conducted to map androgen-related networks and
pathways to improve understanding of the means of transition
and to better distinguish between aggressive and indolent
cancerous tissue [3].
Currently, a three-dimensional cell culture system is being
developed to study cell differentiation and carncinogenesis
[3]. The system utilizes epithelial progenitor cells together
with factors derived from prostatic stromal mesenchyme cells
and extra-cellular matrix to attain gland-like structures [3].
Perturbations in gene expression can then be used to
determine genes involved with differentiation and
transformation. Analysis of the effects of the perturbations
will help define the molecular interactions between epithelial
cell and stromal mesenchyme cells and potential causes of
cancer [3].
Other tools that are being utilized for improving
understanding of tumour distinction include: monoclonal
antibody production, microarray gene expression analysis,
comparative proteomics using mass spectrometry, SNP
detection, mRNA expression, and animal models [3].
3.2 Protease and Tumour Growth
Proteins encoded by the serine protease gene family are
protein-cleaving enzymes, which play a critical role in a wide
range of normal and pathological processes. Included in this
family are several members that play an important role in the
study of normal and neoplastic prostate development [4].
Three of the members in particular, PSA, kK2, and
prostase/PRSS18, are relevant to expression related to the
prostate and are regulated by androgenic hormones. Tissue
extraction of PSA has contributed to early diagnosis and
monitoring of patients with prostate cancer [4]. Several
studies have also been dedicated to regulation of expression of
PSA, identification of its activators and substrates and identify
the different molecular forms of PSA [4]. This research has
improved understanding of the pathophysiology of the disease
and helped improve treatment strategies.
Prostate metastatic involvement has also been more clearly
understood by focussing on the roles of serine proteases.
When they are expressed in non-primary locations, the local
environment may be manipulated by their enzymatic activity
to favour metastasis or tumour growth [4]. It has been shown
that PSA directly degrades extracellular matrix glycoproteins
and assist in cell migration. PSA has been shown to exhibit
properties of androgen-regulated expression. Therefore, it has
been a useful molecule in studies related to the development
of androgen-independent prostate cancer growth. hK2 is able
to activate a protease that has been associated with prostate
cancer metastasis [4].
Serine protease TMPRSS2 was originally cloned from
chromosome 21 using exon-trapping strategies. Its expression
is dependent on androgen and is vastly expressed in normal
and neoplastic prostate epithelium, relative to other human
tissues.
The TMPRSS2 gene encodes a protein of an estimated 492
amino acids, however its exact function is not known [4].
Fairly high expressions have been reported for the prostate,
and weaker expressions have been detected in several other
tissues such as the lung, kidney, and pancreas [4]. Its
activators and substrates have yet to be determined. It could
be autocatalytic or could be activated by other trypsin-like
proteases in the prostate, such as kK2. There is evidence
indicating that it may be a natural activator of the precursor
forms of PSA and hK2. It has been suggested that the
enzymatic activities of TMPRSS2 may have an effect on the
process of prostate carcinogenesis [4].
The projected cell surface expression of TMPRSS2 indicates
that it plays an important role in carcinogenesis and may be
utilized in diagnosis and treatment strategies for prostate
cancers [4]. Further, it is possible that membrane-bound
TMPRSS2 could be cleaved to attain a circulating form.
3.3 Effects of Endostatin on Tumour Migration
The formation of new capillaries from pre-existing blood
vessels is referred to angiogenesis. It is plays a vital role in
certain pathological processes, including inflammation and
tumour growth. As a new therapy for cancer and other
angiogenesis-dependant diseases, anti-angiogenic therapy has
been proposed [5]. Currently, one of the most powerful
inhibitors of angiogenesis is endostatin, which has been
shown to induce tumour regression in mice. Endostatin is
generated by cleavage of collagen by cathepsin L, matrilysin,
or elatase [5]. It has several distinct antiangiogen and tumour
reduction characteristics. Rat endostatin has been shown to
inhibit breast tumour growth [5]. It is also effective using
delivery approaches, such as DNA vaccination or viral
expression under proper scenarios at the site of action.
Endostatin has been shown to block vacular endothelial
growth factor (VEGF)-mediated signalling through KDR/Flk1 [5]. It also stimulates endothelial cell death, associated with
associated with reduced levels of anti-apoptotic proteins Bcl-2
and BclXL. The cell binding effects of endostatin may be a
result of suppression of integrin function [5]. The means of
antiangiogenic and anti-tumour effects in vivo have not yet
been well established. This is partially because it has been
used in both soluble and insoluble forms.
Tumour growth inhibition has been reported with in both
forms, however the regression of tumours has only been
demonstrated using it in the insoluble form [5]. The
bioactivity of endostatin is dependent on its structural form.
Therefore, it is possible that each form may have a distinct
mechanism of producing antiangiogenic effects. It has
recently been discovered that endostatin is a protein with high
tendency to form form amyloid fibers through extensive cross sheet formation [5]. In addition, only insoluble endostatin
stimulates tissue plasminogen activator (t-PA)-regulated
plasminogen activation and leads to cell toxicity [5].
Recent studies have shown that insoluble endostatin:
stimulates plasmin formation; binds plasminogen and t-PA;
stimulates t-PA-mediated plasminogen activation by
endothelial cells; and causes endothelial cell detachment and
extracellular matrix degradation [5]. The formation of
plasmin is associated with degradation of the extracellular
matrix occurring by the prevention of blood clots, tissue
remodelling, migration of cancer cells, and angiogenesis [5].
Several studies have revealed a high expression of t-PA in
several tumours, which is regarded as good prognosis in
cancer patients. By binding t-PA, therapeutic of insoluble
endostatin may stimulate t-PA activity that is produced in the
tumour [5]. Stimulation of the plasmagen can result in
excessive
matrix
degradation,
endothelial
cell
detachment/destruction, inhibition of cell adhesion, or
deterioration of capillary tubes [5]. Endostatin’s stimulation
of plasminogen t-PA-mediated activation indicates that there
may exist a common antiangiogenic pathway that is induced
by t-PA-binding proteins [5].
There is considerable evidence that increased plasminogen
activation may be responsible for at least a portion of the
endostatin effects on tumour growth [5]. In its ability to
overstimulate t-PA, the excessive matrix degradation caused
by endostatin inhibits the onset of angiogenesis and tumour
development [5].
3.4 The Role of Epidermal Growth Factor Receptor (EGFR)
There is considerable evidence indicating that receptor tyrosin
kinases are involved in development and progression of
tumours [6].
The activity of epidermal growth factor
receptor (EGFR) has been correlated to cell migration and
metastasis [6]. Cytoplasmic tyrosine is activated by the
binding of EGFR or transforming gowth factor  (TGF-) to
extracellular domain of EGFR [6]. The cytoplasmic tyrosine
then undergoes autophosphorylation and assembles
downstream effectors to reduce migration. In addition, high
expression of EGFR has been correlated to reduced adhesion
to matrix proteins [6]. This indicates that, in addition to its
role in growth stimulation, EGFR plays a considerable role in
cell migration and invasion.
Increased levels of EGFR have been detected in human
tumours and cell lines, including breast cancer, gliomas, colon
cancer, and bladder cancer [6]. Further, its overexpression has
been associated with invasion and metastases in several
patients.
Studies have indicated that early stages of cell migration are
dependent on mitogen-activated protein kinase (MAPK) and
that later stages are dependent on protein kinase C (PKC)
isoform, PKC--mediated pathways [6]. When combined,
however, MAPK and PKC inhibitors are able to block TGF-induced cell migrations in EGFR-overexpressing breast
cancer [6]. There is evidence that EGFR-overexpressing
invasive cells have the ability to counteract the loss of MAPKmediated signalling through activation of PKC- signalling
for cell migration, which is critical in invasion and metastasis
[6]. Data also indicates that inhibition of MAPK and PKC-
signalling pathways should eliminate cell migration and
invation in EGFR-overexpressing human breast cancer cells
[6].
3.5 Influence of Tetrathiomolybdate on Metastasis
Tetrathiomolybdate (TM) is a specific copper chelator, which
has been shown to be a strong antiangiogenic and metastatic
inhibitor. This is possibly through suppression of the NFB
signalling cascade. The growth and metastatic potential of
tumours with and without the presence of SUM149 has been
significantly inhibited with systemic TM treatment. In
addition, nuclear proteins isolated from TM-treated SUM149
tumours had less NFB binding activity.
Therefore,
suppression of NFkB is likely the primary mechanism by
which TM acts to inhibit angiogenesis and metastasis [7].
3.6 Growth Factors and Organ Specificity
There is evidence supporting the concept that organ specificity
of metastases is influenced by seed and soil compatibility, as
well as physiological factors, such as blood flow and relative
size of cancer cells in comparison to diameter of the
capillaries [8]. Several hypotheses have been proposed
regarding the organ specificity of metastasis. The first of
which postulates that tumour cells that migrate into secondary
sites can survive and reproduce only in those organs that have
appropriate growth factors. Another hypothesis suggests that
chemoattractants home the cancer cells toward specific organs
by means of concentration gradients. A third hypothesis
proposes that secondary tumours can develop in certain
organs, and the endothelial cells of which express adhesion
molecules that can attract the cancer cells. In each hypothesis,
it is suggested that the interaction between cancer cells and
microenvironment affects the direction of metastatic organs
[8].
Fibroblast growth factor receptor 1 (FGFR1) is a receptor for
fibroblast growth factors (FGFs) [8]. FGFR1’s downstream
signals influence mitogenesis and differentiation.
The
microenvironment of bone is likely to be suitable for survival
and emergence of cancer cells that express FGFR1, since
FGFs are expressed in large quantities in bone tissue. There is
an active antagonist that can inhibit bone formation, known as
FST [8]. FST may also promote bone absorption caused by
metastatic cells and contribute to the release of growth factors
stored in bone tissue. This unique relationship between
tumour cells and bone microenvironment appears to be
influential for developing bone metastasis.
The onset of metastasis appears to be dependent on adhesion,
detachment, and aggregation of tumour cells and the adhesive
interface between cancer cells and endothelium is probably
linked to the organ selectivity of metastasis [8].
4.0 CONCLUSION
The purpose of this study was to investigate the current
methods of study of the cancer and the diagnosis and
treatment of tumour growth and metastasis by means of
systems biology technology.
The systems biology approach has become one of the
preferred methods of studying the pathology of cancer. The
development of libraries of structural and functional
information of cancerous tissue has begun to facilitate a
comprehensive understanding of cancer development, growth,
and migration. A more comprehensive understanding of the
genetic mechanism of prostate cancer development will be
facilitated by further development of cell-type-specific cDNA
libraries.
Research in this area has made improved methods of
distinction between aggressive and indolent tumours and the
role of factors such as endostatin, EGRF, TM, and other
growth factors have become more clearly understood.
As research in this area continues and the technology
associated with systems biology continues to develop, a better
understanding of cancer diagnosis, treatment options, and
control will hopefully reduce the severity and number of cases
of cancer worldwide.
5.0 REFERENCES
[1] American Cancer Society, 2003. ACS :: Detailed Guide.
http://www.cancer.org/docroot/CRI/CRI_2_3x.asp?dt=72
[2] Ideker, T., Galitski, T., Hood., L., 2001. A New Approach
to Decoding Life: Systems Biology. Annual Review
Genomics -- Human Genetics 2: 343-372
[3] Institute for Systems Biology. Nanosystems Biology
Alliance and Cancer.
http://systemsbiology.org/Default.aspx?pagename=nanocance
r
[4] Lin, B., Ferguson, C., White, J.T., Wang, S., Vessella, R.,
True, L.D., Hood, L., Nelson, P.S., 1999. Prostate-localized
and Androgen-regulated Expression of the Membrane-bound
Serine Protease TMPRSS2. Cancer Research 59(17): 41804184.
[5] Reijerkerk, A., Mosnier, L.O., Kranenburg, O., Bouma,
B.N., Carmeliet, P., Drixler, T., Meijers, J.C.M., Voest, E.E.,
Gebbink., M.F.B.G., 2003. Amyloid Endostatin Induces
Endothelial Cell Detachment by Stimulation of the
Plasminogen Activation System. Molecular Cancer Research
1(8): 561-568
[6] Kruger, J.S., Reddy, K.B., 2003. Distinct Mechanisms
Mediate the Initial and Sustained Phases of Cell Migration in
Epidermal Growth Factor Receptor-Overexpressing Cells.
Molecular Cancer Research 1(11): 801-809.
[7] Pan, Q., Bao, L.W., Merajver, S.D., 2003.
Tetrathiomolybdate Inhibits Angiogenesis and Metastasis
Through Suppression of the NFB Signaling Cascade.
Molecular Cancer Research 1(10): 701-706.
[8] Kakiuchi, S., Daigo, Y., Tsundoda, Yano., S., Sone, S.,
Nakamura.,
Y.,
Genome-Wide
Analysis
of
Organ0Preferenctial Metastasis of Human Small Cell Lung
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