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GSK3βand β-Catenin Modulate
Radiation Cytotoxicity in
Pancreatic Cancer
1
Richard L. Watson*
,2
, Aaron C. Spalding*
,†,2
,
Steven P. Zielske*, Meredith Morgan*,
Alex C. Kim
‡
, Guido T. Bommer
§,¶
,
Hagit Eldar-Finkelman
#
, Thomas Giordano**,
Eric R. Fearon
‡,§,¶,#
,GaryD.Hammer
‡
,
Theodore S. Lawrence*and Edgar Ben-Josef*
*Department of Radiation Oncology, University of
Michigan Medical School, Ann Arbor, MI, USA;
†
The Norton
Cancer Institute Radiation Center and Kosair Children’s
Hospital, Louisville, KY, USA;
‡
Department of Internal
Medicine—Metabolism, Endocrinology & Diabetes,
University of Michigan Medical School, Ann Arbor, MI, USA;
§
Department of Internal Medicine, University of Michigan
Medical School, Ann Arbor, MI, USA;
¶
Department of
Internal Medicine—Molecular Medicine and Genetics,
University of Michigan Medical School, Ann Arbor, MI, USA;
#
Department of Human Genetics, Tel Aviv University, Ramat
Aviv, Israel; **Department of Pathology, University of
Michigan Medical School, Ann Arbor, MI, USA
Abstract
BACKGROUND: Knowledge of factors and mechanisms contributing to the inherent radioresistance of pancreatic cancer may
improve cancer treatment. Irradiation inhibits glycogen synthase kinase 3β(GSK3β) by phosphorylation at serine 9. In turn, release
of cytosolic membrane β-catenin with subsequent nuclear translocation promotes survival. Both GSK3βand β-catenin have been
implicated in cancer cell proliferation and resistance to death. METHODS: We investigated pancreatic cancer cell survival after
radiation in vitro and in vivo, with a particular focus on the role of the function of the GSK3β/β-catenin axis. RESULTS: Lithium
chloride, RNAi-medicated silencing of GSK3β, or the expression of a kinase dead mutant GSK3βresulted in radioresistance of
Panc1 and BxPC3 pancreatic cancer cells. Conversely, ectopic expression of a constitutively active form of GSK3βresulted in radio-
sensitization of Panc1 cells. GSK3βsilencing increased radiation-induced β-catenin target gene expression as measured by studies
of AXIN2 and LEF1 transcript levels. Western blot analysis of total and phosphorylated levels of GSK3βand β-catenin showed that
GSK3βinhibition resulted in stabilization of β-catenin. Xenografts of both BxPC3 and Panc1 with targeted silencing of GSK3β
exhibited radioresistance in vivo.Silencingofβ-catenin resulted in radiosensitization, whereas a nondegradable β-catenin construct
induced radioresistance. CONCLUSIONS: These data support the hypothesis that GSK3βmodulates the cellular response to
radiation in a β-catenin–dependent mechanism. Further understanding of this pathway may enhance the development of clinical
trials combining drugs inhibiting β-catenin activation with radiation and chemotherapy in locally advanced pancreatic cancer.
Neoplasia (2010) 12, 357–365
Address all correspondence to: Aaron C. Spalding, MD, PhD, The Norton Cancer Institute Radiation Center, 4001 Dutchmans Ln, Louisville, KY 40207.
E-mail: acspalding1@gmail.com
1
These studies were supported by an American Society of Therapeutic Radiology and Oncology Resident Seed Grant (A.S.), and National Institutes of Health R03 CA127050-01
(E.B.J.). These studies were conducted in partial fulfillment for the requirements (R.W.) for graduationwith Honors, College of Literature, Science, and the Arts, from the University of
Michigan. A.Spalding has been designated a B. Leonard Holman Pathway Fellow by the American Board of Radiology, and this work was presented in part at the 2008Gastrointestinal
Malignancies Symposium.
2
These authors contributed equally to this work.
Received 30 December 2009; Revised 15 February 2010; Accepted 16 February 2010
Copyright © 2010 Neoplasia Press, Inc. All rights reserved 1522-8002/10/$25.00
DOI 10.1593/neo.92112
www.neoplasia.com
Volume 12 Number 5 May 2010 pp. 357–365 357
Introduction
Roughly 37,000 US patients are diagnosed annually with pancreatic
cancer, with the annual death rate from pancreatic cancer approaching
its incidence. Aggressive treatment of locally advanced pancreatic can-
cer patients with highly conformal radiation and chemotherapy pro-
duces a moderate beneficial effect on local control of the cancer, albeit
with possible increases in toxicity and a minor impact on patient sur-
vival. Unfortunately, the addition of agents targeting either Ras [1]
(zarnestra, tipifarnib), matrix metalloproteases [2] (marimistat), or
EGFR [3] (erlotinib) has not dramatically increased overall survival
either. Thus, elucidation of the mechanisms underlying pancreatic
cancer radioresistance may lead to improved targeted therapies, which
may improve clinical outcomes.
Depending on the context and cell type under study, glycogen
synthase kinase 3β(GSK3β) can promote cell survival or apoptosis
after cytotoxic insults [4]. Active GSK3βcan induce mitochondrial
release of cytochrome c, leading to the activation of the intrinsic apop-
tosis pathway [5]. Active GSK3βphosphorylates β-catenin, a tran-
scription factor involved in survival and proliferation, at serine 33,
which primes β-catenin for ubiquitination and subsequent proteosome-
mediated degradation [6]. Survival signals such as binding of Wnt ligands
or growth factors to their respective receptors leads to inhibition of GSK3β
through phosphorylation at serine 9. Inhibition of GSK3βthen leads to
the stabilization of β-catenin resulting in nuclear translocation and hetero-
dimerization of β-catenin with T-cell factor family members thus pro-
moting transcription of β-catenin target genes and cell survival [7].
We previously conducted a study that showed that the protein kinase
Cβinhibitor enzastaurin prevents radiation-induced phosphorylation
of protein kinase Cβand leads to radiosensitization through persistent
activation of GSK3βin pancreatic cancer cells. In addition, we demon-
strated that radiation induces phosphorylation of GSK3βat serine 9, a
site known to inhibit GSK3βactivity [8,9]. Our current study expands
on that observation to test the hypothesis that GSK3βmediates the
radiation resistance of pancreatic cancer by suppression of β-catenin.
Our findings establish that the GSK3β/β-catenin pathway modulates
radiation resistance of pancreatic cancer and suggest potential targets
to increase efficacy of radiation therapy in pancreatic cancer.
Materials and Methods
Cell Line Generation
Panc-1 and BxPc3 human pancreatic cancer cells were obtained from
the American Type Culture Collection (Manassas, VA) and were main-
tained according to standard tissue culture conditions. We generated
lentivirus particles for transduction of shRNA to silence GSK3βor
β-catenin. Mission short hairpin RNA (shRNA) lentiviral plasmids
(Sigma, St Louis, MO) contain a U6 promoter transcribing nonspecific
(NS), GSK3β,orβ-catenin shRNA along with a puromyocin resistance
gene for selection. We collected supernatants after cotransfection of
HEK293T cells with mission shRNA and packaging plasmids [10].
BxPC3 or Panc1 cells were then transduced with NS, GSK3β,orβ-
catenin shRNA particles and selected under 2 μg/ml puromycin.
We used lentivirus transduction to express kinase inactive GSK3β
[11]. We subcloned the GSK3β
KK(85,86)MA
KKMA insert using KpnI
(5′end) and XbaI(3′end) from the pCMV4 vector to pLentilox-
IRES-GFP. The resulting pLL−GSK3β
KK(85,86)MA-IGFP
lentiviral
plasmid uses a CMV promoter to drive expression of a single messenger
RNA with both insert and GFP for the identification of transduced
cells. We collected supernatants after cotransfection of HEK293T cells
with empty vector control or pLL−GSK3β
KK(85,86)MA-IGFP
and pack-
aging plasmids. BxPC3 or Panc1 cells were thentransduced with empty
vector control or pLL−GSK3β
KK(85,86)MA-IGFP
particles and analyzed
by flow cytometry. Only cell populations with more than 90% GFP
expression were used.
We used stable transfection to generate cells expressing nondegrad-
able β-catenin [12]. We subcloned the β-catenin
S33Y-FLAG
insert using
BamHI (5′end) and XbaI(3′end) from the pCMV4 vector to pcDNA3.1
(+). Cells were transfected with 1 μg of empty vector control or pcDNA3.1
(+)β-catenin
S33Y-FLAG
and then selected with G418. Stable pooled pop-
ulations of individual clones were verified by Western blot analysis
for FLAG.
Colony Formation Assays
After irradiation, cells were trypsinized, counted, and plated at pre-
determined clonal densities. Two weeks later, cells were fixed with a
methanol/acetic acid mixture (7:1) and stained with crystal violet.
Colony counting was done using an automated counter. Data were
then analyzed by determining the surviving fraction at each dose of
radiation. Cell survival curves were fit using the linear-quadratic
equation. Radiation sensitivity is expressed in mean inactivation dose
(MID), which represents the area under the cell survival curve [13].
MID was calculated for control and each experimental manipulation,
and the enhancement ratio was calculated as the MID in the control
curve divided by the MID in the experimental curve.
Reverse Transcription–Polymerase Chain Reaction
A Qiagen RNeasy RNA extraction kit was used to collect RNA for
reverse transcription–polymerase chain reaction (RT-PCR). RT-PCR
was performed in duplicate using a Qiagen Quantitect Syber Green RT-
PCR kit on GAPDH[14], AXIN2 [15], and Lef1 [16]using previously
published primer sequences. C
T
values for each unknown were com-
pared with a standard curve made of serially diluted RNA from wild-
type BxPC3 and Panc1 cells in logarithmic phase growth. AXIN2 and
Lef1 values were normalized to the level of GAPDH in each sample.
Antibodies and Immunoblot Analysis
Antibodies to GSK3β(Cell Signaling, Danvers, MA), phospho-
Ser9 GSK3β(Cell Signaling), β-catenin (Cell Signaling), phospho-
Ser33 β-catenin (Cell Signaling), and β-actin (Sigma) were used at
dilutions per the manufacturer. Cell lysate production with RIPA buffer
and immunoblot analysis were performed using detailed protocols from
Cell Signaling. Xenograph samples were taken after treatment and
frozen in a dry ice bath. A mortar and pestle was then used to grind
the xenograph samples. β-Actin was used as a control to show that total
protein quantities were equal among the groups. Each Western blot was
performed three independent times from unique lysates; representative
films are shown in Figures 1A,2A,and3A.
Xenografts
Animals used in this study were maintained in facilities approved by
the American Association for Accreditation of Laboratory Animal Care
in accordance with current regulations and standards of the United
States Department of Agriculture and Department of Health and Hu-
man Services. Under an institutionally approved protocol, 4-week-old
female athymic nude mice were implanted with 5 × 10
7
BxPC3 or
358 Wnt Pathway Modulates Radiosensitivity Watson et al. Neoplasia Vol. 12, No. 5, 2010
Panc1 cells subcutaneously. Tumor volume (TV) was calculated accord-
ing to the following equation: TV = Π/6 × a×b
2
,whereaand bare the
longer and shorter dimensions of the tumor, respectively. When the
average tumor volume achieved 100 mm
3
,micewererandomizedto
treatment groups.
Irradiation
Cells or xenografts were irradiated using a Phillips 250 orthovoltage
unit at approximately 2 Gy/min for cells or 1.4 Gy/min for mice in the
Irradiation Core of the University of Michigan Cancer Center. Dosim-
etry is carried out using an ionization chamber connected to an elec-
trometer system, which is directly traceable to a National Institute of
Standards and Technology calibration. Mice were anesthetized with a
mixture of ketamine 60 mg/kg and xylazine 3 mg/kg and positioned
such that the apex of each flank tumor was at the center of a 2.4-cm
aperture in the secondary collimator and irradiated with the rest of
the mouse being shielded from radiation.
Statistical Analysis
The clonogenic assays were conducted on three independent occa-
sions in triplicate. Mean and SD from the three independent experi-
ments are displayed in Figures 1A,2,Band C, and 6. A two-tailed
t-test was used to analyze differences between mean values of in vitro
assays, with αvalues less than 0.05 considered significant. The radia-
tion enhancement factor (REF) was calculated as previously described
[17], with numbers less than 1 indicating radioprotection and numbers
greater than 1 indicating radiosensitization.
The RT-PCR data in Figure 5Arepresent the mean and SD values
of three independent experiments performed in triplicate after irradi-
ation. A two-tailed t-test was used to analyze differences between
mean values at each time point, with αvalues less than 0.05 consid-
ered significant.
The in vivo experiments were designed with a power of 80% to
detect a 20% difference in tumor growth delay between the control
versus irradiated tumors, resulting in a sample size of 10 tumors per
group. Tumor volumes are plotted relative to the pretreatment volume
in Figure 3, Band C. A two-tailed t-test was used to analyze differences
between mean values at each measurement, with αvalues less than 0.05
considered significant.
Results
GSK3βSignaling Modulates Radiation Resistance In Vitro
Inhibition of GSK3βby phosphorylation at Ser9 has been pre-
viously observed after irradiation of pancreatic cancer cells [17], po-
tentially underscoring their observed radioresistance. We examined
the phenotypic effects of GSK3βmodulation on radiation response
in vitro. Previous studies have shown that lithium chloride (LiCl) is a
pharmacological inhibitor of GSK3β, with inhibition correlating with
increased phosphorylation of GSK3βat serine 9 [18]. We determined
the concentration of LiCl needed to increase GSK3βphosphorylation
and found that 30 mM was associated with phosphorylation in BxPC3
and Panc1 cells (Figure 1A). Inhibition of GSK3βby a 6-hour exposure
to LiCl before radiation led to an increase in survival in response to
radiation in both BxPC3 and Panc1 cells (REFs: 0.78 and 0.79, respec-
tively, P< .05; Figure 1B). Because pharmacologic inhibition such as
LiCl treatment may have unintended off-target effects, we also used
genetic approaches to test our hypothesis that GSK3βinhibition pro-
motes radioresistance in pancreatic cancer.
To further characterize the role of GSK3βin radiation survival, we
transduced BxPC3 and Panc1 cells with a lentivirus construct expressing
Figure 1. (A) BxPC3 and Panc1 cells were treated with LiCl for 6 hours, and Western blot analysis for total and phosphorylated GSK3β
was performed. (B) Clonogenic survival of control (○) or LiCl-pretreated (•) BxPC3 and Panc1 cells. *P≤0.05. Error bars are SD of three
independent experiments performed in triplicate and are smaller than the symbols at some data points.
Neoplasia Vol. 12, No. 5, 2010 Wnt Pathway Modulates Radiosensitivity Watson et al. 359
an shRNA designed to inhibit GSK3βexpression. We generated poly-
clonal populations of BxPC3 and Panc1 cells expressing the shRNA
construct and then determined the effect of GSK3βknockdown on
survival after radiation. Radiation delivered to pancreatic cancer cells ex-
pressing an NS shRNA construct resulted in serine 9 phosphorylation,
similar to wild-type cells. Ser9 GSK3βphosphorylation was increased
with a peak at 1 hour after a 2-Gy radiation (Figure 2A). Silencing of
GSK3βprevented radiation-induced GSK3βserine phosphorylation in
response to a 2-Gy radiation and produced radioresistance (REFs of
0.82 in BxPC3 and 0.60 in Panc1, P< .05; Figure 2B) similarly to
pharmacological inhibition of GSK3β. These data indicate that inhibi-
tion of GSK3βpromotes survival in response to irradiation.
As a second genetic approach, we generated polyclonal populations
of cells stably expressing GSK3β
KK(85,86)MA
, which has an inactive
substrate phosphorylation domain. Expression of the kinase dead
GSK3β
KK(85,86)MA
inhibited radiation cytotoxicity compared with
cells transduced with empty vector (REFs of 0.76 in BxPC3 and
0.70 in Panc1; Figure 2C). These data show that radiation resistance
of pancreatic cancer cells in vitro can be modulated through manipu-
lation of GSK3β.
GSK3βSignaling Modulates Radiation Resistance In Vivo
After observing the radioprotective effect of GSK3βinhibition in vitro,
we studied the consequences of GSK3βinhibition in vivo. Polyclonal
populations of BxPC3 and Panc1 cells expressing GSK3βshRNA
maintained knockdown of GSK3 10 weeks after subcutaneous implan-
tation, whereas those with NS shRNA retained the expression of
GSK3β(Figure 3A). Control BxPC3 xenografts expressing NS shRNA
exhibited a 26-day growth delay with ten 2-Gy fractions (Figure 3B)
and a 61-day growth delay with ten 3-Gy fractions (Figure 3C). Si-
lencing of GSK3βleads to shortened growth delay from both the 2-Gy
(17 days, REF of 0.65) and the 3-Gy (25 days, REF of 0.40) treatment
courses (P< .05 for both). Similarly, control Panc1 xenografts expressing
NS shRNA exhibited a 24-day growth delay with five 2-Gy fractions
Figure 2. (A) BxPC3 and Panc1 cells expressing NS or GSK3βshRNA were treated with 2 Gy, and Western blot analysis for total and
phosphorylated GSK3βwas performed. The blots were confirmed in at least three independent experiments. (B) Clonogenic survival of
NS (○) or GSK3βshRNA (•) BxPC3 and Panc1 cells. (C) Clonogenic survival of empty vector control (○) or GSK3β
KK(85,86)MA
(•) BxPC3 and
Panc1 cells. *P≤0.05. Error bars are SD of three independent experiments performed in triplicate and are smaller than the symbols at
some data points.
360 Wnt Pathway Modulates Radiosensitivity Watson et al. Neoplasia Vol. 12, No. 5, 2010
(Figure 3D) and a 43-day growth delay with five 3-Gy fractions
(Figure 3E). Silencing of GSK3βleads to shortened growth delay from
the 2-Gy (16 days, REF of 0.64) and the 3-Gy (23 days, REF of 0.53)
treatment courses (P< .05 for both). Thus, tumors without GSK3βwere
less sensitive to radiation, similar to the results from the in vitro clono-
genic assays.
To determine changes in vivo induced by radiation, a separate ex-
periment with identical arms was conducted; tumors were collected
immediately after the last fraction of radiation, and staining for hema-
toxylin and eosin (H&E) and Ki67 was performed (Figure 4). H&E
staining revealed that knock down of GSK3βresulted in increased
nuclear-to-cytoplasmic ratio and decreased production of mucin, sug-
gesting cellular dedifferentiation, a phenotype consistent with β-catenin
activation. Radiation reduced the proliferation index from 95% to 30%
in NS shRNA tumors, whereas GSK3βshRNA tumors had a less pro-
nounced reduction from 98% to 65% (Figure 4B).
The decreased tumor growth delay, increased tumor cell density,
and increased proliferation in the GSK3βknockdown tumors all cor-
relate with the in vitro observation that inhibition of GSK3βpro-
motes radiation resistance.
Modulation of the Radiation Response through β-Catenin
We hypothesized that GSK3βmodulates the radiation response
through a β-catenin–dependent gene transcription. We first tested
whether radiation induced β-catenin activity. In BxPC3 and Panc1 cells
expressing NS shRNA, radiation induced the transcription of Lef1 and
Axin2 [19], two well-characterized β-catenin target genes, in a time-
dependent manner as measured by quantitative RT-PCR. Targeted
Figure 3. (A) Xenografts from BxPC3 and Panc1 cells expressing NS or GSK3βshRNA were analyzed for expression of GSK3β. The blots
were confirmed in at least three independent experiments. BxPC3 NS shRNA and GSK3βknockdown xenografts were treated with ten
2-Gy fractions (B) or ten 3-Gy fractions (C) and were compared with unirradiated controls. Panc1 NS shRNA and GSK3βknockdown
xenografts were treated with five 2-Gy fractions (D) or five 3-Gy fractions (E) and were compared with unirradiated controls. *P≤
0.05 between the NS versus GSK3βknockdown. Error bars are SEM of the 10 tumors per treatment arm. The dashed line indicates
a four-fold increase in tumor size, used to determine the enhancement ratio.
Neoplasia Vol. 12, No. 5, 2010 Wnt Pathway Modulates Radiosensitivity Watson et al. 361
silencing of GSK3βresulted in both higher basal and radiation-induced
levels of Lef1 or Axin2 gene transcription (Figure 5A). Because we ob-
served that radiation affects β-catenin transcriptional activity in vitro
through GSK3β, we hypothesized that radiation would have similar
effects in vivo (Figure 5B). Before irradiation, β-catenin localized to the
cytosolic membrane. After radiation of xenografts, β-catenin translocated
to the nucleus, suggesting induction of β-catenin signaling. Radiation
induction of β-catenin nuclear translocation correlates with the obser-
vation in vitro that GSK3βphosphorylation modulates β-catenin–
dependent gene transcription.
If GSK3βmodulates pancreatic cancer cell response to radiation
through β-catenin, then modulation of β-catenin activity may influ-
ence cell survival after radiation. Therefore, we transduced BxPC3
and Panc1 cells with lentivirus encoding shRNA targeting β-catenin.
Compared with cells transduced with NS shRNA, cells with silenced
β-catenin were more sensitive to radiation as shown by reduced clono-
genic survival (REFs of 1.4 in BxPC3 and 1.25 in Panc1; Figure 6).
On the basis of these experiments, constitutive activation of β-
catenin would be predicted to render pancreatic cancer cells resistant
to radiation. Therefore, a β-catenin
S33Y-FLAG
vector was used to create
cells expressing constituently active β-catenin. The S33Y mutation
prevents GSK3β-mediated phosphorylation at Ser33, thus preventing
ubiquitination and subsequent degradation [20]. Cells expressing con-
stituently active β-catenin
S33Y
showed increased clonogenic survival
(REF for Panc1 cells was 0.8; Figure 6C). The effects of nondegradable
β-catenin
S33Y
were analogous to those resulting from GSK3βinhibition
or silencing because both showed an increased resistance to radiation
together with an increased level of β-catenin activity. Thus, increased
β-catenin activity results in greater radiation resistance of pancreatic
cancer cells, whereas loss of β-catenin through RNAi-mediated silenc-
ing results in increased radiation sensitivity.
Discussion
In this study, we found that inhibition of GSK3β, by either genetic
or pharmacological methods, induces radiation resistance of pancre-
atic cancer cells in vitro, reduces the duration of radiation-induced
tumor growth delay, and leads to increased cell proliferation in vivo.
Similarly, the expression of a constituently active β-catenin in pancreatic
cancer cells increases the resistance to radiation. Our results reinforce
and expand on previous studies of radiation effects on GSK3β.
We have previously demonstrated that radiation induces phos-
phorylation of GSK3βat Ser9, an event known to inhibit GSK3β
kinase activity [17], and that abrogation of this phosphorylation re-
sulted in radiosensitization. Radiation was also shown to inhibit
GSK3βactivity in SAOS-2 cells [21], although the phenotypic con-
sequences in sensitivity to radiation were not investigated. Irradiation
of A549 cells induced phosphorylation of GSK3βat Ser9, and this
effect was reduced when cells were plated on fibronectin [22]. The
authors suggested that GSK3βis involved in the interaction of cells
with the extracellular matrix after radiation to modulate the cytotox-
icity of radiation. These studies implicate GSK3βas a mediator of
radiation sensitivity.
We hypothesized that GSK3βmodulates radiation cytotoxicity, at
least in part, through its downstream effector β-catenin. Herein, we
show that radiation induces the transcription of Lef1 and Axin2, two
well-characterized β-catenin target genes, and targeted silencing of
GSK3βresults in both higher basal and radiation-induced levels of
Figure 4. Xenografts from BxPC3 and Panc1 cells expressing NS or GSK3βshRNA were analyzed by H&E (A). Black arrows indicate
glandular structures present in the NS shRNA xenografts, which are absent in GSK3βshRNA xenografts. Panc1 xenografts with and
without radiation were analyzed for proliferation by Ki67 (B). Original magnification, ×400.
362 Wnt Pathway Modulates Radiosensitivity Watson et al. Neoplasia Vol. 12, No. 5, 2010
Lef1 or Axin2 gene transcription. Furthermore, we show that radia-
tion induces translocation of cytosolic β-catenin to the nucleus in
Panc1 and BxPC3 xenographs, an observation consistent with the
in vitro induction of transcription of Lef1 and Axin2. Finally, we show
that cells with silenced β-catenin are more sensitive, whereas cells expres-
sing constituently active β-catenin
S33Y
are more resistant to radia-
tion. β-Catenin has been shown to prevent epithelial cell death
after radiation or anoikis [23]. These findings suggest that β-catenin
is involved in determining clonogenic survival of pancreatic cancer
cells after irradiation.
Our studies potentially explain the relationship between Wnt sig-
naling and radiation cytotoxicity in other tumor sites. Activation of the
Wnt signaling pathway resulted in β-catenin cytoplasmic accumulation
with translocation to the nucleus in head and neck cancer cell lines
expressing COX-2 [24]. In turn, up-regulation of Ku expression leads
to increased radioresistance. Blocking COX-2 signaling led to the sup-
pression of β-catenin–induced Ku expression and consequent radiation
sensitivity. Others have suggested that the radioresistance observed clin-
ically in glioblastoma depends in part on the activation of β-catenin in
putative cancer stem cells [25]. In a mouse model of breast develop-
ment, radiation selectively enriched for mammary epithelial progenitors
isolated from transgenic mice with activated Wnt/β-catenin signaling
but not for background-matched controls [26]. We conversely showed
that suppressing β-catenin using shRNA correlated with an increase in
radiation sensitivity.
Our data reinforce observations from others that GSK3βinhibition
protects normal tissue from radiation toxicity. Radiation-induced
GSK3βactivation results in mouse hippocampal neuronal apoptosis
and subsequent neurocognitive decline. The expression of kinase-inactive
GSK3βor pharmacologic inhibition before irradiation significantly
attenuated radiation-induced apoptosis in hippocampal neurons, lead-
ing to improved cognitive function in irradiated animals [27]. Mice
treated with lithium chloride, a known GSK3βinhibitor, had decreased
neurocognitive impairment after irradiation as well [28]. Akt serves to
inhibit GSK3βafter irradiation in normal vascular endothelium [29],
and administration of recombinant growth factors known to activate
Figure 5. (A) Time course of Lef1 and Axin2 levels in NS (○) or GSK3βshRNA (•) BxPC3 and Panc1 cells subjected to 2-Gy radiation.
Mean of three experiments with SDs, *P≤0.05. (B) BxPC3 or Panc1 xenografts were treated with 2-Gy radiation and were stained for
β-catenin (green) and propidium idodide (red). Yellow indicates overlap of red and green, consistent with nuclear β-catenin.
Neoplasia Vol. 12, No. 5, 2010 Wnt Pathway Modulates Radiosensitivity Watson et al. 363
Akt may prevent normal tissue toxicity. However, any pharmacologic
strategy to reduce normal tissue damage must be carefully weighed
against the risk of tumor protection.
Our results are consistent with radioprotection caused by active
β-catenin. A reporter mouse model demonstrated that ionizing radia-
tion activates β-catenin–mediated, T-cell factor–dependent transcrip-
tion both in vitro and in vivo. Mouse-derived fibroblast cultures
expressing stabilized β-catenin formed more colony-forming units than
wild-type or null cells after irradiation. β-Catenin levels in irradiated
wounds correlated with tensile strength of the wound, and lithium
chloride treatment also increased β-catenin levels and increased wound
strength [30]. The newly identified R-Spondin1 augments canonical
Wnt/β-catenin signaling and causes nuclear translocation of β-catenin.
R-Spondin1 reduced mucosal ulceration after whole-body or snout-
only irradiation in mouse models [31]. Therefore, in normal cells, GSK3β
inhibition with β-catenin activation may be a radioprotective mecha-
nism. Pancreatic cancer cells potentially invoke a similar mechanism
to evade the cytotoxic effects of radiation.
Our results help explain an apparent contradiction present in the
literature regarding pancreatic cancer and β-catenin. Mutations in
APC leading to β-catenin nuclear accumulation have been well char-
acterized to play a role in colon cancer. However, mutations in APC
[32] or β-catenin [33] have not been found in pancreatic cancer. The
published literature suggests that constitutive activation of β-catenin
does not play a role in pancreatic cancer development. In fact, our
results also demonstrate similar findings, as unirradiated tumors
lacked nuclear β-catenin, and we did not find evidence of increased
β-catenin target gene expression without irradiation. However, we
did find that pancreatic cancer cells activate β-catenin in response
to radiation to promote survival. Our results may therefore explain
in part the clinically observed radioresistance of pancreatic cancer;
specifically, it may not be the basal level of β-catenin but rather the in-
duction of β-catenin by radiation that promotes pancreatic cancer cell
survival. We plan to test this hypothesis by immunoflorescence of pan-
creatic cancer specimens treated with neoadjuvant radiation to deter-
mine whether activation of β-catenin occurs in patients.
The implications of this work identify a link between radiation
and a pathway central to tumor growth, invasion, and metastasis
of pancreatic cancer. By further discovering the molecular signaling
cascades upstream and downstream of GSK3β, we will also start to
gain insight into the potential interactions with other signaling path-
ways that are known to be involved in radioresistance. Further under-
standing of this pathway will also help develop clinical trials combining
drugs inhibiting β-catenin activation with radiation and cytotoxic
agents in locally advanced pancreatic cancer.
Acknowledgment
The authors thank Steven Kronenberg for his graphical expertise.
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