Molecular Tailoring of Chemotherapy with Novel
Molecular Beacons, Gene Silencing, and Reporter Genes Studied
to Better Predict Response to Chemotherapy
(Philadelphia, PA) – Researchers at the Abramson Cancer
Center of the University of Pennsylvania are applying a host
of imaging techniques to develop better ways to look noninvasively at
the molecular characteristics of tumors. The experiments, now in human
cell cultures and mouse models, are aimed at better forecasting early
response to chemotherapy so that treatment choices can be adjusted.
“Right now in cancer therapy, with the exception of relatively uncommon
examples of cancers for which we have tumor markers, we don’t have
reliable ways of predicting who is going to respond early on to chemotherapy,”
says Wafik El-Deiry, MD, PhD, Associate Professor, Departments
of Medicine, Genetics, and Pharmacology. “Currently cancer patients
get their chemo and you can’t tell if they’re responding for
several weeks. We need to have tests that will tell us if patients are
going to respond to the chemo or the radiation soon after it’s first
given, and whether these responses are going to last.”
Two recent papers in Cancer Biology & Therapy and Cancer
Research describe the work of the El-Deiry laboratory. One approach
is to use a molecular beacon, a molecule that can be activated within
cells due to a specific context, such as in this case, the response to
chemotherapy. The beacon recognizes a characteristic change in chemo-treated
tumor cells, physically opens up and fluoresces, which can then be measured.
“The beacon goes right into the living cell and if it opens up,
emitting fluorescence, we can detect the glow,” says El-Deiry.
Human lung-cancer cells were treated with the chemotherapeutic agent doxorubicin
(Adriamycin), which causes cellular DNA damage. Doxorubicin works through
the tumor suppressor protein p53, which ultimately kills many types of
cancer cells. “We engineered a molecular beacon to detect expression
of a gene called p21, that is turned on directly by p53 when cells are
exposed to Doxorubicin,” says El-Deiry.
cells that were exposed to Doxorubicin activated the p53-responsive molecular-beacon
tag and emitted a strong fluorescence. (Click on thumbnail to view full-size
schematic). From this El-Deiry and colleagues hope to develop a scan that
could detect a patient’s likely reaction to certain chemotherapies:
Strong fluorescence equals a good response to the chemotherapy. They hope
to make what he refers to as a “beacon cocktail” that can
predict response by monitoring multiple genes simultaneously as well as
additional intracellular events in the process of cell death.
In another study El-Deiry and colleagues combine imaging techniques and
a mouse model for colon cancer. “In this research, we’re combining
two very powerful emerging technologies,” says El-Deiry. “This
is the first example, to my knowledge, of the use of inducible gene silencing
and non-invasive bioluminescence imaging in a mouse model for cancer.”
Gene silencing is a technique that allows researchers to control the expression
of any gene in a given cell by introducing small RNA sequences targeting
the gene of interest. Inducible refers to the ability to control whether
or not the silencing RNA is expressed in the cell so that investigators
can compare gene activity to tumor growth, as El-Deiry did in this study.
This approach allows researchers to regulate gene expression by what they
feed the mice. In this study, the KILLER/DR5 receptor, another protein
that responds to chemotherapy by killing cancer cells, is silenced in
colon tumors in the mice.
They also labeled the cells with a reporter gene called firefly luciferase,
which gives off light. “The use of a reporter like firefly luciferase
marks the tumor cells so we can see them by another imaging technique,”
explains El-Deiry. “Fireflies that we see in the evening carry out
the same chemical reaction with their own luciferase protein to give off
the light.” The imager detects the light and captures its intensity
to provide a measurement of the size of the tumor.
bioluminescence imaging technology has provided a breakthrough that allows
scientists to examine the size of a tumor in living mice with high sensitivity,”
says El-Deiry. (Click on thumbnail to view full-size image). “Since
the reporter gene is always on and only in the tumor cell, it’s
essentially measuring tumor volume. Using the reporter gene along with
the KILLER/DR5 silencer, we show for the first time that when we turn
off KILLER/DR5, we get bigger tumors.”
While the beacon or beacon cocktails have the potential to be used in
the clinic to detect mutations in cancer cells or the activation of genes
that predict therapeutic response, the major advance with the bioluminescence
imaging is in accelerating preclinical drug development. The gene silencing
allows precise molecular characterization of targets that are relevant
for therapeutic response while the imaging allows non-invasive assessment
of drug activity towards implanted tumors. This approach saves time and
money because it is possible to see the effects of drugs in living mice
without sacrificing them and it also requires fewer mice in experiments.
Because the KILLER/DR5 receptor is involved in the process of cell death
by chemotherapy, El-Deiry is also gaining insight into which drugs use
it and which drugs work by other mechanisms. “This is important
because to maximize tumor killing and to attempt to bypass or reverse
resistance to chemotherapy, we need to harness all the ways cancer cells
can be killed,” he says.
The KILLER/DR5 receptor is engaged by a therapeutic agent currently being
developed called TRAIL (Tumor necrosis factor-Related Apoptosis Inducing
Ligand). TRAIL is produced normally by natural killer cells and controls
tumor spread by binding to a tumor’s death-inducing receptor KILLER/DR5.
“However, in cancer patients with suppressed immunity and for reasons
we still don’t understand, there isn’t enough TRAIL being
produced or effectively delivered by the natural killer cells at the site
of tumors and so tumors are not suppressed,” says El-Deiry. “The
hope is that if TRAIL is administered to patients alone or in combination
with chemotherapy, this may in the clinic lead to some benefit.”
TRAIL looks promising in animal studies but clinical studies that are
due to start in the next year or so will determine how toxic TRAIL is
and begin to see whether it really works in cancer patients.
The work was funded by NIH grants including a multi-institutional Network
for Translational Research in Optical Imaging imaging grant from the National
Cancer Institute. The Network provides support for imaging resources to
accelerate translational research on cancer.
The Abramson Cancer Center of the University of Pennsylvania
was established in 1973 as a center of excellence in cancer research,
patient care, education and outreach. Today, the Abramson Cancer Center
ranks as one of the nation’s best in cancer care, according to U.S.
News & World Report, and is one of the top five in National Cancer
Institute (NCI) funding. It is one of only 39 NCI-designated comprehensive
cancer centers in the United States. Home to one of the largest clinical
and research programs in the world, the Abramson Cancer Center of the
University of Pennsylvania has 275 active cancer researchers and 250 Penn
physicians involved in cancer prevention, diagnosis and treatment.
PENN Medicine is a $2.7 billion enterprise dedicated
to the related missions of medical education, biomedical research, and
high-quality patient care. PENN Medicine consists of the University of
Pennsylvania School of Medicine (founded in 1765 as the nation’s
first medical school) and the University of Pennsylvania Health System
(created in 1993 as the nation’s first integrated academic health
Penn’s School of Medicine is ranked #3 in the nation for receipt
of NIH research funds; and ranked #4 in the nation in U.S. News &
World Report’s most recent ranking of top research-oriented medical
schools. Supporting 1,400 fulltime faculty and 700 students, the School
of Medicine is recognized worldwide for its superior education and training
of the next generation of physician-scientists and leaders of academic
Penn Health System is comprised of: its flagship hospital, the Hospital
of the University of Pennsylvania, consistently rated one of the nation’s
“Honor Roll” hospitals by U.S. News & World Report; Pennsylvania
Hospital, the nation's first hospital; Presbyterian Medical Center; a
faculty practice plan; a primary-care provider network; two multispecialty
satellite facilities; and home health care and hospice.