Powerpoint template for scientific posters Swarthmore College

1
Dissecting the Role of p53 in Tumor Response to
Radiation Therapy with Primary Lung Cancers in
Mice
Bradford A. Perez, A. Paiman Ghafoori, Samuel M.
Johnston, Laura B. Jeffords, Yongbaek Kim,
Cristian T. Badea, David G. Kirsch Duke
University School of Medicine, Durham, NC
Discussion We have developed a medium-high
throughput method to ask important questions
about lung cancer biology using advanced mouse
models of primary lung cancer. On arrival in the
laboratory, serial imaging of more than
approximately 5 mice with lung tumors in one day
was not possible. Now, we are able to image as
many as 60 mice in one day which allows us to
perform important experiments in a primary tumor
model system. Our system is superior to
traditional xenograft or even knockout mouse
models because tumors form within the expected
tumor environment (lung parenchyma) and
tumorigenesis is temporally controlled by the
initiation of tumorigenesis with Adeno-Cre
infection during adulthood. Furthermore,
expression of mutant alleles driving
tumorigenesis occurs at reasonable physiologic
levels because mutant alleles are inserted at the
site of and under the control of endogenous gene
promoters. It is our feeling that this
preclinical model recapitulates the human tumor
experience in the mouse with much greater
accuracy than traditional models. We look
forward to having the opportunity to better
understand tumor biology by altering important
genes in the tumor and surrounding
microenvironment using Cre-lox or other similar
technologies. We have future plans to test new
innovative treatment strategies while closely
monitoring response to therapy by Micro-CT using
our advanced mouse models and current serial
imaging workflow. In this study which aimed to
understand the role of p53 in the response to
radiation treatment, we have shown that our
LSL-K-rasG12D ink4a/ARFflox/flox model maintains
the ability to activate p53 and its downstream
effectors p21 and puma. We have also shown
functional activation of known p53 dependent cell
cycle arrest response in the LSL-K-rasG12D
ink4a/ARFflox/flox model. Conversely, our mouse
in which tumorigenesis is partially driven by a
deletion mutation in p53 (LSL-K-rasG12D
p53flox/flox ) does not seem to be able to
initiate a cell cycle arrest response following
radiation treatment. Our in vivo studies, which
are functional tumor growth delay assays, show
that both models respond well to radiation
treatment. Interestingly, our model with intact
p53 function seems to have an even better
response to 2 radiation treatments delivered 24
hours apart despite the fact that the doses
delivered with each treatment strategy were
calculated to be biologically equivalent. This
improved response to multiple radiation
treatments has not been appreciated in our tumor
model in which p53 undergoes a deletion mutation
that drives tumorigenesis. Potential mechanisms
for this improved response in tumors with intact
p53 function are interesting and have important
clinical implications. It is possible that
activation of p53 cell cycle arrest in the tumor
cells following radiation treatment helps to
synchronize tumor cells so that at the time of
the next fractionated radiation treatment dose a
greater percentage of the cells are in a more
radiosensitive phase of the cell cycle such as
G2/M. Another possibility is that something
inherent to deletion mutation at the Ink4a/ARF
locus is the reason for improved response to
fractionated radiation treatment. If we can
identify characteristics of patient tumors that
would benefit from hyper-fractionated radiation
therapy it may be reasonable to consider altering
standard treatment paradigms in these patients to
improve outcomes. Alternatively, if we
understand that a patients tumor will not
benefit from increased fractionation, we can
offer hypofractionated regimen such as
stereotactic body radiotherapy which is often
more convenient. We look forward to performing
future, more in depth, comprehensive studies to
investigate the mechanism for improved response
to fractionated radiation therapy in our tumor
model that maintains wild-type p53
(LSL-K-rasG12D ink4a/ARFflox/flox ) as we work
to understand why this effect is not appreciated
in our tumor model that is initiated and
maintained by a p53 deletion mutation
(LSL-K-rasG12D p53flox/flox ).
Introduction Many studies have been performed to
elucidate the role of p53 in radiation response
at the molecular level. Most experts agree that
cells respond to DNA double strand breaks caused
by high energy radiation via recruitment of the
protein Ataxia-Telangectasia mutated (ATM) and
related proteins known as PI3-kinase like protein
kinases (PIKKs). Once activated, ATM has been
shown to quickly phosphorylate a large number of
additional proteins including p53, MDM2, and CHK2
proteins. Many of the proteins phosphorylated by
ATM directly or indirectly lead to stabilization
and activation of the p53 protein. Activation of
p53 leads to many different downstream events.
p53 activation generally causes either cell cycle
arrest or senescence which can slow growth, or
apoptosis leading to cell death. The type of
response conferred by p53 activation seems to be
cell type dependent. Traditionally, it has been
thought that loss of normal p53 function leads to
treatment resistance because tumor cells are less
likely to undergo apoptosis. However, this is
only likely to be true in tumors that originate
from tissues in which apoptosis is the primary
response following activation of p53. Among
tumors that originate from tissues in which
apoptosis is not the primary p53 dependent
response, it is unclear to what extent p53 plays
a role in sensitivity to radiation treatment.
Often in these tumor cell types cell cycle arrest
occurs which can allow time for DNA repair and
may be protective during radiation treatment.
Alternatively, p53 may cause tumor cells to enter
a permanent, non-cycling, senescence state. In
the absence of p53, tumor cells may be less
likely to undergo apoptosis or cell cycle arrest.
When the primary response for a particular cell
type is apoptosis, p53 inactivation likely leads
to radiation resistance. However, when the
primary cell type response is cell cycle arrest,
loss of p53 may lead to increased radiation
sensitivity because accumulation of DNA damage
can be so great that cells are unable to complete
mitosis and undergo a type of cell death known as
mitotic catastrophe.
Results
Experimental Design
F
A
C
Puma expression
p21 expression
p53 function intact
no p53 function
LSL-K-rasG12D ink4a/ARFflox/flox
LSL-K-rasG12D p53flox/flox
Relative Fold Change
Relative Fold Change
Week 1
A
LSL-K-rasG12D ink4a/ARFflox/flox
LSL-KrasG12D p53flox/flox
Infect mice with Adeno-cre intranasally
D
G
Week 8
BRDU immunostaining
Tumor Growth Delay In Vivo
Image mice using Micro-CT
Week 10
Image mice using Micro-CT
B
Irradiate mice with 0, 1 ,or 2 radiation
treatments
p53 expression
LSL- KrasG12D Ink4a/ArfFL/FL
LSL- KrasG12D p53FL/FL
With this project, we set out to learn more about
what role p53 may be playing in the response of
lung tumors to radiation treatment. We compared
two different primary tumor mouse models of
NSCLC. While the tumors grew at the same
baseline rate, one model retained wild-type p53
expression while in the other tumor model
tumorigenesis was driven by p53 deletion
mutation.
E
H
Tumor Growth Delay In Vivo
BRDU Quantification
Week 12
Perform Immunohistochemical studies to determine
mechanism of p53 response
Image Mice using Micro-CT
Relative Fold Change
B
Dark-before radiation Light-4hrs after 11.6 Gy
Treatment
What is Cre-Lox technology?
Results -A-Confirmation that tumors with
LSL-K-rasG12D ink4a/ARFflox/flox and
LSL-K-rasG12D p53flox/flox have similar
histological features (above)and grew at
approximately the same baseline rate (below).
B-Quantitative RT PCR performed on RNA isolated
from tumors with LSL-K-rasG12D
ink4a/ARFflox/flox (blue, n11) and
LSL-K-rasG12D p53flox/flox (red, n11) tumors
confirms decreased p53 gene expression in the
LSL-K-rasG12D p53flox/flox cohort (p0.0001).
C-A similar experiment strongly suggests
increased p21 expression following radiation
treatment in the LSL-K-rasG12D
ink4a/ARFflox/flox cohort which maintains
wild-type p53 function (p0.06,4 hrs. timepoint).
D,E-BRDU staining confirms functional cell cycle
arrest in tumors with wild-type p53
(LSL-K-rasG12D ink4a/ARFflox/flox )after
radiation treatment when compared to tumors with
a mutation deletion in p53 (LSL-K-rasG12D
p53lfox/flox) (p0.004). F- Further quantitative
RT PCR confirms increased activation of p53
downstream effector, PUMA, in tumors with intact
p53 function (p0.04, 4 hrs. timepoint).
G-LSL-K-rasG12D ink4a/ARFflox/flox tumors
perform better after both 1 and 2 radiation
treatments calculated to deliver biologically
equivalent doses. Importantly 2 treatments
confers a statistically significant treatment
advantage when compared to single dose treatment
(p0.001). H-LSL-K-rasG12D p53flox/flox tumors
also show growth delay after 1 and 2 radiation
treatments, however 2 treatments does not confer
significant treatment advantage when compared to
single dose treatment (p0.21). .

Cre is a site-specific DNA recombinase that
actively targets sites known as loxP sequences.
Recombination of two direct repeat loxP sites on
the same chromosome causes a deletion event which
removes any sequence between LoxP sites.
C
Day 1
Day 14
Day 28
Experimental Design A-Schematic documenting
custom built micro-CT scanner with mouse cradle
at Duke Center for In Vivo Microscopy. Images
collected are reconstructed using software (Cobra
Exxim) with a resolution of 88 microns/pixel and
then analyzed for new tumors and tumor growth.
SC-spinal cord, H-heart B-Radiation treatment
set up. Mice were safely and securely placed in
cradles with lead blocks in place on either side
of the tissue to protect normal tissue.
C-Serially reconstructed Micro-CT scan documents
clear and measurable tumor growth over time.

• Relevant Literature
• Jackson, E. L., K. P. Olive, et al. (2005). "The
  differential effects of mutant p53 alleles on
  advanced murine lung cancer." Cancer Res 65(22)
  10280-8.
• Jackson, E. L., N. Willis, et al. (2001).
  "Analysis of lung tumor initiation and
  progression using conditional expression of
  oncogenic K-ras." Genes Dev 15(24) 3243-8
• Aguirre AJ, et al. (2003). Activated Kras and
  Ink4a/Arf deficiency cooperate to produce
  metastatic pancreatic ductal adenocarcinoma.
  Genes Dev. 17(24)3112-26.
• Badea, C. T., M. Drangova, et al. (2008). "In
  vivo small-animal imaging using micro-CT and
  digital subtraction angiography." Phys Med Biol
  53(19) R319-50.
• Sharpless, N. E. and R. A. Depinho (2006). "The
  mighty mouse genetically engineered mouse models
  in cancer drug development." Nat Rev Drug Discov
  5(9) 741-54.
• Gudkov AV, Komarova EA. (2003) The role of p53
  in determining sensitivity to radiotherapy. Nat
  Rev Cancer. 3(2)117-29.
• Lane, D. P. (1992). "Cancer. p53, guardian of the
  genome." Nature 358(6381) 15-6.
• Vousden, K. H. and X. Lu (2002). "Live or let
  die the cell's response to p53." Nat Rev Cancer
  2(8) 594-604
• Kemp, C. J., S. Sun, et al. (2001). "p53
  induction and apoptosis in response to radio- and
  chemotherapy in vivo is tumor-type-dependent."
  Cancer Res 61(1) 327-32.

Acknowledgements I would like to thank
Chang-Lung Lee, Jeffrey Mito, and Dr. Rebecca
Dodd for insightful conversations related to this
project. I would also like to thank Drs. Al
Johnson and Larry Hedlund for their support and
discussions regarding mouse imaging at the Center
for In Vivo Microscopy. This work was supported
with medical student research grants from the
HHMI and the RSNA.