Download The Relationship Between Epidermal Growth Factor Receptor

Predicting Cervical Carcinoma Cell Response to
Epidermal Growth Factor Receptor Inhibition
Adam Searleman1 Raysha Crawford2 and Craig D. Woodworth3
Department of Biology, Clarkson University
Cervical cancer is the second most common cancer in women, and is linked to many
cancer-related deaths each year, particularly in developing countries. The major risk factor for
cervical cancer is infection with a subset of high-risk human papillomaviruses (HPV), in
particular HPV types 16 or 18. However, most HPV infections of the cervix require additional
cofactors for progression to full malignancy. One important cofactor is the epidermal growth
factor receptor (EGF-R). The EGF-R is a member of a family of four closely related
transmembrane tyrosine kinases, each of which can dimerize with other members of the family
upon the binding of a ligand. Upon ligand binding, the EGF-R autophosphorylates on
intracellular tyrosine residues which initiate signal transduction cascades that ultimately result in
altered gene expression. In this fashion, the EGF-R can stimulate cell proliferation, angiogenesis,
and cell motility, as well as inhibit cell differentiation and programmed cell death. These are all
functions that can contribute to cancer progression.
The EGF-R and the closely related receptor ErbB2 are overexpressed in many human
cancers, including cervical carcinoma, and overexpression of the EGF-R has been linked to a
poor prognosis and decreased survival rates. Since the EGF-R has an important role in
progression to cervical cancer, it is a promising target for anticancer therapy. Clinical testing of
various antibodies and tyrosine kinase inhibitors has shown that this treatment is effective,
particularly when combined with traditional cancer treatments such as chemotherapy or radiation.
However, there is significant variability in the response of different cancers to EGF-R inhibitors,
and some patients receive little benefit from these drugs. The purpose of this study is to determine
how a specific cervical cancer cell line will respond to EGF-R inhibition by PD153035.
PD153035 is a small molecule tyrosine kinase inhibitor that specifically blocks activity of the
EGF-R on its intracellular domain. Previous research suggests that there is no correlation
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2
3
Adam Searleman, Bio-Molecular Science and Mathematics Major, class of 2006
Raysha Crawford, Biology Major, Monroe Community College and NIH 2+2 Summer Program
Associate Professor, Department of Biology, Clarkson University
between the level of EGF-R expression and sensitivity to EGF-R inhibition. One explanation
may be that cell lines that are resistant to EGF-R inhibition have mutations downstream, resulting
in less dependence on EGF-R for stimulating tumorigenic cell responses. However, the response
of the cervical cancer cells to PD153035 was analyzed by determining how PD153035 inhibited
cell growth, and there is another valid response to consider.
Traditional cancer therapy uses either cytotoxic agents or ionizing radiation to effect
apoptosis, the body’s natural defense mechanism against mutation. Cancerous cells are generally
more susceptible to apoptosis than normal cells. Drugs that target critical pathways for tumor
development can sensitize resistant cancer cells to traditional cancer therapy. This also prevents
non-specific drug interactions with normal cells, which reduces the severity of side-effects. EGFR inhibiting drugs have been shown to have such a synergistic effect in conjunction with
radiation and chemotherapy. In the studies described here, fifteen cervical cells lines which were
previously characterized for their growth inhibition by PD153035 were also investigated for the
effect of PD153035 on their response to the proapoptotic agents etoposide and hydrogen
peroxide. Etoposide induces apoptosis by inhibiting topoisomerase II, an enzyme that maintains
DNA structural integrity during replication. Cervical cells are sometimes naturally exposed to
hydrogen peroxide, which can induce apoptosis by oxidative stress. The cervical cell lines were
exposed to varying doses of etoposide or hydrogen peroxide to find a dose that induced apoptosis
in approximately 20% of the cells (AD20). This dose was then used with varying concentrations
of PD153035 to determine how effectively PD153035 sensitizes each cell line to apoptosis
initiated by two unique mechanisms.
The level of expression of ErbB2, the preferred binding partner of EGF-R, may be an
important predictor of response to EGF-R inhibition. Alternatively, response might be correlated
to the amount of phosphorylated, or activated, EGF-R. Lysates were previously extracted from
the 15 cervical cancer cell lines using RIPA buffer supplemented with appropriate protease
inhibitors. A sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) was
used to separate the proteins, which were subsequently transferred to a nitrocellulose filter. The
level of expression of either ErbB2 or phosphorylated EGF-R was detected by immunoblotting
with polyclonal antibodies and visualized by chemiluminescence.
The levels of expression of each ligand that activates EGF-R are also possible predictors.
Overexpression of ligands such as EGF, TGF-α, HB-EGF, betacellulin, amphiregulin, and
epiregulin may induce hyperactive EGF-R stimulation. The level of expression of these ligands
is being investigated by reverse transcriptase polymerase chain reaction (RT-PCR). The RNA of
10 cell lines was previously extracted, reverse-transcribed into cDNA and then amplified by PCR.
The amplified cDNAs were then separated by an agarose gel and visualized using ethidium
bromide. The preliminary results indicate that several ligands are expressed equally in all cell
lines and other ligands have very poor correlations to growth inhibition.
The long-term goal of this project is to predict how different cancers respond to EGF-R
inhibition. An improved understanding of this response might help physicians to decide whether
to treat a cancer patient with drugs that inhibit EGF-R function or with other conventional
therapies.