Vol 9 No1 - Journal of Cell and Molecular Biology - Haliç Üniversitesi

84 Necip Ozan TİRYAKİOĞLU et al.
Introduction
Epidermal growth factor receptor (EGFR) is a cell
surface receptor that activates signalling cascades
which mainly induce proliferation and cell survival.
Human genome codes for four members of the
EGFR family, EGFR/ErbB1, HER2/ErbB2/neu-2,
HER3/ErbB3 and HER4/ErbB4. All the family
members consists of a ligand-binding, a
transmembrane and a cytoplasmic domain with
tyrosine kinase activity (Gschwind et al., 2004).
EGFR members reside on the cell membrane as
monomers which dimerize upon phosphorylation
by ligand binding. Eight different molecules have
been identified as EGFR/ErbB1 ligands. All the
EGFR ligands, except Crypto, are synthesized as
precursor molecules and become functional only
after proteolytic clevage. In addition, EGFR family
members have been shown to be activated
indirectly by chemokines, cell adhesion molecules
and G-protein coupled receptors (Yarden and
Sliwkowski, 2001). The dimerisation of EGFR
members can occur as homodimerisation or as
heterodimerisation with another member of the
family. Following dimerisation,
autophosphorylation in homodimers and
transphosphorylation in heterodimers occurs.
During this process five tyrosine residues, Y992,
Y1045, Y1068,Y1148 and Y1173, located on Cterminal
are phosphorylated. Consequently, EGFRs
activate a multitude of transducer and adapter
proteins including HP-2, GRB2, PI3K and Akt.
Activation of these proteins induce a variety of
cytoplasmic pathways, mainly MAPK and JNK,
which trigger cellular events like DNA synthesis
and proliferation (Oda et al., 2005).
Dysregulation of EGFRs are mainly caused by
overexpression of the receptor itself, their ligands
or both. In addition to overexpression, mutations in
the tyrosine kinase domain causing consistent
activation of the receptor account for another major
mechanism affecting EGFR signalisation. Such
mutations have been identified in lung, stomach
and breast cancer tissues, but not in normal tissues.
Dysregulation of EGFR can also be the result of
HER2/ErB2 heterodimerisation. HER2 does not
have ligand binding activity, instead it increases
the ligand binding affinity of the protein it
dimerises with. Thus, EGFR/HER2 dimerisation
leads to a more active receptor and increased
activity of the EGFR pathways (Herbst, 2004).
Upon discovery of disturbed EGFR functions in
multiple cancers, abundant number of studies have
been conducted in order to develop drugs targeting
EGFR. Inhibitory molecules targeting EGFR can be
classified in three main categories; monoclonal
antibodies, chemical inhibitors and antisense
oligonucleotides.
Monoclonal antibodies exert their inhibitory
effect by binding to the ligand binding domain and
disrupt activation upon ligand binding. Monoclonal
antibodies against EGFR have been showed to
inhibit cell growth and induce apoptosis. However,
since they exert their effect only on ligand binding
domain, EGFRs with tyrosine domain mutations
are prone to be irresponsive to the inhibitory effects
of monoclonal antibodies. In contrast to
monoclonal antibodies, chemical inhibitors target
tyrosine kinase domain and inhibit EGFR by
blocking ATP from interacting with the tyrosine
kinase domain. Antisense nucleotides, on the other
hand, prevent translation of the EGFRs and their
ligands and carry out their inhibitory effect at a
much essential level.
90% of the somatic EGFR mutations are located
in the tyrosine kinase domain, namely exons 18-21
(Table 1). Since tumor cells depend on the
activating mutations on tyrosine kinase domain of
EGFR for proliferative signals, it has been shown
that tumor cells harboring such mutations are more
sensitive to thymidine kinase inhibitors (TKI).
Thus, determination of the EGFR mutations has
both prognostic value as an indicator of drug
response and diagnostic value as a biomarker
(Lynch et al. , 2004).
Since the determination of EGFR mutations
requires tumour samples and collecting a tumour
sample is only possible through invasive
techniques, EGFR mutation surveys are limited to
tissue availability. Being able to scan for the EGFR
mutations by using whole blood as an alternative
DNA source may provide information about the
genotype of the original tumour tissue without the
need for invasive techniques. Therefore, the study
presented here is initiated as the first step of a wider
research with the final goal of matching mutations
in tumour tissues with that of normal somatic cells
from the same individuals.
Table 1. Common EGFR Mutations
Mutations Frequency Effect
G719A/C/S 3% Increased sensitivity
Ex 19 Deletion 48% Increased sensitivity
Ex20 Deletion 4% Decreased sensitivity
T790M 5% Decreased sensitivity
L858R 43% Increased sensitivity
L861Q 2% Increased sensitivity