Article

Inhibition of Mammalian Target of Rapamycin or Apoptotic Pathway Induces Autophagy and Radiosensitizes PTEN Null Prostate Cancer Cells

Vanderbilt University, Нашвилл, Michigan, United States
Cancer Research (Impact Factor: 9.33). 11/2006; 66(20):10040-7. DOI: 10.1158/0008-5472.CAN-06-0802
Source: PubMed
ABSTRACT
The phosphatidylinositol 3-kinase/Akt pathway plays a critical role in oncogenesis, and dysregulation of this pathway through loss of PTEN suppression is a particularly common phenomenon in aggressive prostate cancers. The mammalian target of rapamycin (mTOR) is a downstream signaling kinase in this pathway, exerting prosurvival influence on cells through the activation of factors involved in protein synthesis. The mTOR inhibitor rapamycin and its derivatives are cytotoxic to a number of cell lines. Recently, mTOR inhibition has also been shown to radiosensitize endothelial and breast cancer cells in vitro. Because radiation is an important modality in the treatment of prostate cancer, we tested the ability of the mTOR inhibitor RAD001 (everolimus) to enhance the cytotoxic effects of radiation on two prostate cancer cell lines, PC-3 and DU145. We found that both cell lines became more vulnerable to irradiation after treatment with RAD001, with the PTEN-deficient PC-3 cell line showing the greater sensitivity. This increased susceptibility to radiation is associated with induction of autophagy. Furthermore, we show that blocking apoptosis with caspase inhibition and Bax/Bak small interfering RNA in these cell lines enhances radiation-induced mortality and induces autophagy. Together, these data highlight the emerging importance of mTOR as a molecular target for therapeutic intervention, and lend support to the idea that nonapoptotic modes of cell death may play a crucial role in improving tumor cell kill.

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Available from: Sekhar Konjeti
Inhibition of Mammalian Target of Rapamycin or Apoptotic
Pathway Induces Autophagy and Radiosensitizes
PTEN Null Prostate Cancer Cells
Carolyn Cao,
1
Ty Subhawong,
1
Jeffrey M. Albert,
1
Kwang Woon Kim,
1
Ling Geng,
1
Konjeti R. Sekhar,
1
Young Jin Gi,
2
and Bo Lu
1
Departments of
1
Radiation Oncology and
2
Surgical Oncology, Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine,
Nashville, Tennessee
Abstract
The phosphatidylinositol 3-kinase/Akt pathway plays a critical
role in oncogenesis, and dysregulation of this pathway
through loss of PTEN suppression is a particularly common
phenomenon in aggressive prostate cancers. The mammalian
target of rapamycin (mTOR) is a downstream signaling kinase
in this pathway, exerting prosurvival influence on cells
through the activation of factors involved in protein synthesis.
The mTOR inhibitor rapamycin and its derivatives are cyto-
toxic to a number of cell lines. Recently, mTOR inhibition has
also been shown to radiosensitize endothelial and breast
cancer cells in vitro. Because radiation is an important
modality in the treatment of prostate cancer, we tested the
ability of the mTOR inhibitor RAD001 (everolimus) to enhance
the cytotoxic effects of radiation on two prostate cancer cell
lines, PC-3 and DU145. We found that both cell lines became
more vulnerable to irradiation after treatment with RAD001,
with the PTEN-deficient PC-3 cell line showing the greater
sensitivity. This increased susceptibility to radiation is
associated with induction of autophagy. Furthermore, we
show that blocking apoptosis with caspase inhibition and
Bax/Bak small interfering RNA in these cell lines enhances
radiation-induced mortality and induces autophagy. Together,
these data highlight the emerging importance of mTOR as a
molecular target for therapeutic intervention, and lend
support to the idea that nonapoptotic modes of cell death
may play a crucial role in improving tumor cell kill. (Cancer
Res 2006; 66(20): 10040-7)
Introduction
Prostate cancer is the most common cancer in men. Radiother-
apy is a mainstay in managing early-stage or inoperable locally
advanced disease. Recent innovations in three-dimensional con-
formal and intensity-modulated radiation therapy have allowed for
maximal delivery of radiation while limiting toxicity (1, 2). Finding
agents that sensitize malignant cells to radiation would increase
tumor response while minimizing toxicity to surrounding organs
by lowering effective therapeutic doses.
The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)
pathway promotes normal cell growth and proliferation (3), and its
constitutive activation has been implicated in many human
cancers, including the pancreas, ovary, and stomach (4, 5).
Although direct amplification of PI3K/Akt has not been found in
prostate cancer (5), mutations in the suppressor of this pathway,
PTEN, have been described with high frequency, especially in more
advanced and aggressive neoplasms (5, 6).
The mammalian target of rapamycin (mTOR), also known as
RAFT1, RAPT1, and FRAP, is a 289 kDa serine-threonine kinase
downstream of Akt (7). mTOR phosphorylates and inactivates the
translation suppressor eukaryotic initiation factor 4E-binding pro-
tein 1 (4E-BP1, PHAS1), and also activates ribosomal p70 S6 kinase
(S6K1). Principally, through these two factors, the normal activation
of mTOR results in an increase in global protein synthesis (8, 9).
Recently, it has been suggested that dysregulation of mTOR contri-
butes to oncogenesis in a broad range of cancers (10). Although
precise mechanisms are unknown, mTOR-mediated alterations in
protein synthesis, aberrant cell cycle signaling, and inhibition of
apoptosis may all play causal roles (11, 12). The ability of mTOR
inhibitors to attenuate progrowth, proproliferative, and prosurvival
actions of mTOR has therefore generated much interest (10).
mTOR presents an attractive therapeutic target in the PI3K/Akt
pathway because it acts downstream of broader function upstream
proteins (13). PI3K inhibitors, such as wortmannin and LY294002,
have limited clinical potential due to lack of kinase specificity and
metabolic instability (14). However, when the immunosuppressant
rapamycin binds its intracellular target FKBP12, this complex
specifically blocks mTOR-mediated phosphorylation of S6K1 and
4E-BP1, providing more effective pathway targeting (9). In vitro
studies have established the ability of rapamycin to inhibit cellular
transformation, and potent rapamycin derivatives, such as CCI-779,
AP 23573, and RAD001, have been developed for use in cancer
therapy ( for review, see ref. 15). Transgenic mice with activation of
Akt or deficiencies in PTEN have shown sensitivity to these agents
(16), and RAD001 specifically has been shown to reverse prostate
neoplastic phenotypes in mice expressing human Akt (17).
Although mTOR inhibitors have been shown to increase the
radiosensitivity of endothelial and breast cancer cells (18–20), their
potential radiosensitizing effects have not been specifically
examined in prostate cancer cells. In this study, we examined the
combined effects of RAD001 and irradiation on cell survival in both
PTEN/ PC-3 and PTEN+/+ DU145 cell lines. Because we found
that mTOR inhibition confers radiosensitivity and induces the
nonapoptotic cell death pathway of autophagy, we also examined
induction of autophagy with apoptosis inhibitors as a way to
enhance radiation cytotoxicity. Because solid tumors are only
variably sensitive to apoptotic death, our results support the notion
that alternatives to apoptosis, such as autophagy, may be exploited
to improve tumor cell killing (21).
Note: C. Cao and T. Subhawong contributed equally to this work.
Requests for reprints: Bo Lu, Department of Radiation Oncology, Vanderbilt
University, B-902 The Vanderbilt Clinic, 1301 22nd Avenue South, Nashville, TN 37232-
5671. Phone: 615-343-9233; Fax: 615-343-3075; E-mail: bo.lu@vanderbilt.edu.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-0802
Cancer Res 2006; 66: (20). October 15, 2006
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Research Article
Page 1
Materials and Methods
Cell culture and drug treatment. PTEN/ PC-3 and PTEN+/+ DU145
prostate carcinoma cells (American Type Culture Collection, Rockville, MD)
were maintained in RPMI 1640 with 10% fetal bovine serum and 1%
penicillin/streptomycin. All cells were incubated at 37jC and humidified 5%
CO
2
. Irradiation was given by use of a
137
Cs irradiator (J.L. Shepherd and
Associates, Glendale, CA). RAD001 was obtained from Novartis Pharma-
ceutical (East Hanover, NJ), Z-VAD from Axxora (San Diego, CA), and Bax
and Bak small interfering RNA (siRNA) from Santa Cruz Biotechnologies
(Santa Cruz, CA).
In vitro clonogenic assay. PC-3 and DU145 cells were treated
with RAD001 (10 nmol/L, 1 hour), Z-VAD (10 Amol/L, overnight), or
DMSO control. Transfection with siRNA Bak (20 nmol/L) and siRNA Bax
(20 nmol/L) or siRNA ATG5 (20 nmol/L) and siRNA Beclin-1 (20 nmol/L)
or MS control (40 nmol/L) was carried out by use of a mixture of 20 AL
LipofectAMINE 2000/mL (Life Technologies, Gaithersburg, MD). Cells
were irradiated with 0 to 6 Gy as indicated, at a dose rate of 1.8 Gy/min.
After irradiation, the medium was changed, and cells were incubated at
37jC for 8 days. Cells were then fixed for 15 minutes with 3:1 methanol/
acetic acid and stained for 15 minutes with 0.5% crystal violet (Sigma-
Aldrich, St. Louis, MO) in methanol. After staining, colonies were visually
counted with a cutoff of 50 viable cells. Surviving fraction was calculated
as (mean colonies counted) / [(cells plated)
(plating efficiency)], where
plating efficiency was defined as (mean colonies counted) / (cells plated) for
nonirradiated controls. Experiments were conducted in triplicate with
mean, SD, and P values (Student’s t test) calculated.
Western immunoblots. Cells were treated (RAD001 50 nmol/L, 1 hour)
or irradiated (5 Gy) according to the individual study. Two hours
postirradiation, cells were washed with ice-cold PBS twice before the
addition of lysis buffer. Equal amounts of protein were loaded into each well
and separated by 6.5%, 10%, or 15% SDS-PAGE gel, followed by transfer onto
nitrocellulose membranes. Membranes were blocked by use of 5% nonfat
dry milk or 1% bovine serum albumin in TBS-T buffer for 1 hour at room
temperature. The blots were then incubated overnight at 4jC with rabbit
anti-phospho-mTOR, total mTOR, phospho-AKT, total AKT, phospho-S6,
and total S6 antibodies, all at 1:1,000 and purchased from Cell Signaling
Technology (Beverly, MA); rabbit anti-h-actin antibody (1:2,000) was
acquired from Sigma-Aldrich. Goat anti-rabbit IgG secondary antibodies
(1:1,000; Santa Cruz Biotechnologies) were incubated for 1 hour at room
temperature. Immunoblots were developed using the enhanced chemilu-
minescence detection system (Amersham, Piscataway, NJ) according to the
protocol of the manufacturer and autoradiography. Band intensity was
quantified using image analysis.
Green fluorescent protein-light chain 3 plasmid transfection. PC-3
and DU145 cells were grown on sterile histologic slides in a square plate
with 15 mL medium, and after 24 hours the cells were transfected with
green fluorescent protein-light chain 3 (GFP-LC3) plasmid (gift from
Dr. Norboru Mizushima, Department of Bioregulation and Metabolism,
Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) by use of
a mixture of LipofectAMINE (Life Technologies) and GFP-LC3 plasmid
in Opti-MEM medium (Life Technologies) at a ratio of 12 AL Lipofect-
AMINE/mL medium per 2 AL plasmid. After 5 hours of incubation, cells
were placed in regular complete media and cultured for one day. RAD001
(10 nmol/L, 1 hour) or Z-VAD (10 Amol/L, overnight) was added. After cells
were irradiated with 3 Gy as indicated, the medium was changed, and cells
were further incubated for 24 hours at 37jC. The slides were taken out and
washed with cold PBS, and cells were fixed in cold methanol for 5 minutes
at room temperature. Cells then were washed in PBS twice, and coverslips
were mounted with glycerol/PBS (3:1) solution. Slides were examined on an
Olympus fluorescent microscope and color print pictures were taken
(
1,000).
siRNA transfection. siRNA Bax (mouse), siRNA Bak (mouse), siRNA Bax
(human), siRNA Bak (human), siRNA ATG5 (human), and siRNA Beclin
(human) were purchased from Santa Cruz Biotechnologies. Cells were
transfected with 25 nmol/L siRNAs using LipofectAMINE 2000. The
transfected cells were used for subsequent experiments 24 hours later.
Measurement of apoptosis. Annexin V-FITC is a sensitive probe for
identifying apoptotic cells. Propidium iodide is a standard flow cytometric
viability probe and is used to distinguish viable from nonviable cells.
Apoptosis was measured using Annexin V-FITC Apoptosis Detection kit I
(BD PharMingen, San Diego, CA) with flow cytometry. PC3 and DU145 cells
were plated into 100 mm dishes for each data point. After 24 hours of 37jC
incubation, the cells were treated with RAD001 for 1 hour. Cells were then
irradiated with 3 Gy as indicated. Medium was then changed and cells were
further incubated for 24 hours at 37jC. Cells were then trypsinized (keeping
all floating cells) and counted. Cells were washed twice with cold PBS and
then resuspended in 1
binding buffer. One hundred microliters of the
solution (5
10
5
cells) were transferred to a culture tube to which 3 AL
Annexin V-FITC and 3 AL propidium iodide were added. After 15 minutes
of incubation at room temperature in the dark, 400 ALof1
binding buffer
was added to each tube and samples were analyzed by FACScan.
Results
Radiosensitization of prostate cancer cells by RAD001. To
determine whether the mTOR inhibitor RAD001 radiosensitizes
prostate cancer cells, we used clonogenic assays to assess cell
survival. First, we examined drug treatment alone and found that
10 nmol/L RAD001 decreased cell survival in both PC-3 cells and
DU145 cells compared with controls (Fig. 1A). PC-3 cells were
more sensitive to mTOR inhibition, as the survival of treated PC-3
cells was 63% that of untreated cells. The survival of treated DU145
cells was 76% that for untreated cells, suggesting a slight relative
resistance of DU145 to drug treatment compared with PC-3 (Fig. 1A).
Based on a previously developed protocol, cells were treated with
RAD001 (10 nmol/L) or DMSO control for 1 hour before irradiation
(18). One hour provides sufficient time for mTOR inhibition to
occur without producing significant cytotoxicity, ensuring that any
changes in cell survival are attributable to RAD001-induced
radiosensitivity (20). Clonogenic assays showed decreased survival
in both cell lines (Fig. 1B). Importantly, RAD001 exerts a more
potent radiosensitizing effect on PC-3 cells compared with DU145
cells, as evidenced by the dose-enhancing ratio (DER) of 1.2 seen in
treated PC-3 cells, compared with 1.02 in DU145 cells (Fig. 1B).
These results suggest that mTOR inhibition with RAD001 enhanced
the radiosensitivity of PC-3 cells by f20%, 10 times higher than
that attained in DU145 cells.
Effect of radiation and RAD001 on mTOR signaling in
prostate cancer cells. To establish the efficacy of RAD001 in
inhibiting mTOR signaling and examine the effects of radiation and
RAD001 combination on mTOR signaling, PC-3 and DU145 cells
were treated with 10 nmol/L RAD001 alone, 5 Gy alone, or in com-
bination. Levels of p-Akt, p-mTOR, and p-S6 (mTOR downstream
target) were measured with Western immunoblots 2 hours after
treatment (Fig. 2). As expected, PTEN-competent DU145 cells
expressed lower levels of p-Akt compared with PC-3 cells at baseline.
Additionally, radiation alone did not increase levels of p-Akt,
p-mTOR, or p-S6. Previous experiments showed that neither cell
line exhibited appreciable postradiation induction of these proteins
at 15 minutes, 30 minutes, or 1 hour postirradiation (data not shown).
Our results showed dramatic attenuation of p-S6 expression after
treatment with RAD001 in PC-3 cells (Fig. 2), confirming the ability of
the drug to block mTOR signaling. Although low levels of baseline
p-S6 were detected in DU145 cells despite the presence of p-mTOR,
RAD001 completely eliminated this limited expression of p-S6. This
indicates total blockade of downstream mTOR signaling in these
cells (Fig. 2). Combination treatment with both RAD001 and radiation
also resulted in the inhibition of phosphorylation of S6, demonstrat-
ing successful attenuation of mTOR activity in both cell lines.
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Radiation induces cell death in prostate cancer cells treated
with RAD001. To elucidate the mechanism responsible for the
RAD001-induced radiosensitization of PC-3 cells, flow cytometry
was used to determine the relative contribution of apoptosis to
increased cell death following irradiation. Figure 3A shows that in
PC3 cells, apoptosis was not up-regulated in response to mTOR
inhibition alone. Additionally, adding RAD001 to irradiated cells
only mildly increased apoptosis above the levels achieved by 3 Gy
alone. Similarly, in DU145 cells, mTOR inhibition alone did not
increase levels of apoptosis, but combination treatment with
radiation resulted in higher levels of apoptosis than that of
radiation alone (Fig. 3A). Importantly, the basal level of apoptosis
observed in untreated cells was demonstrably higher in the DU145
cell line, reinforcing the assumption that unchecked PI3K/Akt
signaling promotes survival in PTEN/ PC-3 cells.
Indirect immunofluorescence with cotransfected GFP-LC3
plasmid was used in PC-3 cells to investigate whether autophagy
was induced as an alternative cell death pathway. Microtubule-
associated protein-1 LC3 is an important component of mamma-
lian autophagosomes, and thus the GFP-LC3 fusion protein has
been used as a reliable marker for their presence (19, 22). In PC-3
cells, the subcellular localization of LC3 changed in response to
radiation from a diffuse scattering throughout the cytosol to a
characteristic punctate pattern indicative of autophagosome
formation (Fig. 3B). As shown in Fig. 3C, quantitative analysis of
this effect revealed that RAD001 alone fails to enhance this process
above baseline, although when combined with radiation it
produces a synergistic increase, with nearly 60% of transfected
cells displaying the signature fluorescence pattern of autophagy.
DU145 cells did show a quantitative increase in autophagosome
formation following treatment with RAD001 alone, as well as with
radiation alone. Furthermore, combining RAD001 with radiation
resulted in the highest level of autophagy.
Blocking apoptosis promotes autophagy and enhances
radiosensitivity in PC3 and DU145 cells. Our unpublished data
suggested that by inhibiting apoptosis, cells can be radiosensitized
through alternative cell death pathways. To further investigate this
phenomenon, we examined the effects of blocking caspase-
dependent apoptosis with Z-VAD on autophagosome formation
and clonogenic survival. Our results show significant increase
of autophagosome formation achieved with 10 Amol/L Z-VAD
(Fig. 4A), with >2-fold increases seen in both cell lines. We then
analyzed both PC3 and DU145 cells with clonogenic assays to
determine the extent to which this effect modulates postradia-
tion cell mortality, and whether mTOR inhibition further enhances
this process. In the PC-3 cell line, the results indicate a large
survival difference between controls and those cells treated with
RAD001, Z-VAD, or a combination of both (Fig. 4B). Treatment
with 10 Amol/L Z-VAD alone yielded a DER of 1.24, and in
Figure 2. RAD001 (50 nmol/L) effectively abrogates p-S6 expression, a marker
for mTOR inhibition, in both cell lines, although the effect is more easily
visualized in PC-3 cells due to their high basal expression of p-S6. Western
analysis confirms low levels of p-Akt in PTEN+/+ DU145 cells. Neither irradiation
nor RAD001 induce PI3K/Akt signaling.
0.00
0.20
0.40
0.60
0.80
DU145
Survival of RAD001 treated cells
relative to control
A
B
0.001
0.01
0.1
1
024 6
Dose (Gy)
Surviving Fraction
Vehicle treated PC-3
cells
RAD001 treated PC-
3 cells
Vehicle treated
DU145 cells
RAD001 treated
DU145 cells
RAD001 treated PC-3 cells DER: 1.20
RAD001 treated DU145 cells DER: 1.02
*
p-value 0.001
*
p-value = 0.022
PC-3
Figure 1. RAD001 sensitizes PTEN/ PC-3 cells to irradiation. A, colony
assay to assess relative cell survival of RAD001 (10 nmol/L,
1 hour) treated to
control PC-3 cells shows that mTOR inhibition results in significant cell killing.
This effect is less pronounced in DU145 cells, as the ratio of cells surviving
treatment to control cells was higher (0.76 versus 0.63). B, clonogenic assays
reveal that PC-3 cells exhibit log-cell killing with increasing doses of radiation;
additionally, mTOR inhibition with RAD001 (10 nmol/L,
1 hour) heightens this
radiosensitivity (P < 0.001). Although DU145 cells do show some increased
radiosensitivity after mTOR inhibition (P = 0.02), the effect is not nearly as
dramatic.
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combination with RAD001 the DER increased to 1.36. In DU145
cells, Z-VAD alone only minimally affected survival with a DER of
1.05, and Z-VAD/RAD001 combination yielded a modest DER of
1.08 (Fig. 4C).
An in vitro Bax/Bak knockout mimic was generated by using
siRNAs against Bax and Bak to confirm the effects of apoptosis
inhibition on radiosensitization. It was determined that 20 nmol/
L each of Bax/Bak siRNA was sufficient to block expression of
these proapoptotic factors in both cell lines (Fig. 5A). Clonogenic
assays then done using this concentration of Bax/Bak siRNA
showed that, similar to Z-VAD, inhibiting Bax/Bak resulted in an
increase in the radiosensitivity of PC-3 cells (DER 1.26; Fig. 5B).
This effect was enhanced when RAD001 was added in combina-
tion treatment (DER 1.35; Fig. 5B). In DU145 cells, Bax/Bak siRNA
induced a smaller increase in postirradiation cell death, resulting
in a DER of 1.14 (Fig. 5C). Again, combination treatment with
both RAD001 and Bax/Bak siRNA resulted in the highest
radiosensitization (DER 1.26). To confirm that the increase in
radiosensitivity was due to an increase in autophagy, we repeated
GFP-LC3 transfection for both cell lines (Fig. 5D). Although
radiation alone and RAD001 alone each increased levels of
autophagy, the most dramatic increase was observed for
combination treatment, with nearly triple the number of trans-
fected cells showing autophagosome formation in PC-3 cells
compared with control (0.694 versus 0.246, respectively) and more
than double in DU145 cells (0.476 versus 0.206, respectively). To
determine whether the observed radiosensitization is partly
dependent on autophagic process, we attenuated autophagy by
transfecting siRNAs against ATG5 and Beclin-1 into the prostate
cancer cells. Figure 5E shows the attenuation of ATG5 and Beclin-
1 levels seen 24 hours after transfection of the respective siRNAs
into the prostate cancer cells. As shown in Fig. 5F, cells
transfected with ATG5 and Beclin-1 siRNA became more resistant
to radiation as their survival curves shifted upwards (DER for
siRNA control versus siRNA ATG5 and Beclin1 is 1.31, P = 0.008
for PC3 cells; and 1.19, P = 0.004 for DU145 cells).
Discussion
In the present study, we found that both mTOR and apoptosis
inhibition sensitize PTEN/ PC-3, but not PTEN+/+ DU145, cells
to the cytotoxic effects of radiation. This is associated with the
promotion of autophagy. Our data did not show increased mTOR
signaling in the prostate cancer cell line DU145 after irradiation;
however, the high basal activity of the unsuppressed Akt pathway
in PTEN/ PC-3 cells make it difficult to show further increases
in mTOR signaling after irradiation. By providing more substrate
on which RAD001 could act, this heightened baseline expression
may account for the difference in how these cell lines responded to
drug treatment.
Radiation-induced activation of the PI3K/Akt/mTOR prosur-
vival pathway is counterproductive to the killing of malignant
Figure 3. Radiation induces increased
cell death in prostate cancer cells treated
with RAD001. A, Annexin V flow cytometry
apoptosis assay shows that mTOR
inhibition alone does not significantly
contribute to apoptotic cell death in PC-3
cells or DU145 cells. However, RAD001
does mildly increase the percentage of
cells undergoing apoptosis postirradiation.
The lower basal level of apoptosis in
PC-3 cells recapitulates the constitutive
activation of PI3K/Akt–mediated
prosurvival signaling in these PTEN /
cells. B, GFP-LC3 transfection studies
show that mTOR inhibition promotes
autophagosome formation in PC-3 and
DU145 cells. B, a representative
photograph (
1,000) in prostate cancer
cells shows that the characteristic punctate
appearance of GFP-LC3, indicative of
autophagosome formation, increases
with both radiation and combination
radiation plus RAD001. C, quantitative
measurement of the percentage of
autophagic cells out of all GFP-transfected
cells shows that although RAD001 alone
induces little autophagy, mTOR inhibition
works synergistically with radiation in
driving cells to autophagy in prostate
cancer cells. Notably, of GFP-transfected
cells, a higher percentage undergo
autophagy at baseline in DU145 cells
compared with PC-3 cells, again reflecting
loss of PTEN suppression of PI3K/Akt
prosurvival signals in PC-3 cells.
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cells, and this induction can occur within 15 minutes after
irradiation (23, 24). Specifically, events following mTOR-mediated
phosphorylation of translational machinery enhance expression of
the protective kinase TLK1B, which confers cellular radioresistance
(25). Indeed, the capacity to achieve radiosensitization through
mTOR inhibition has been shown before (18–20). Our results
similarly indicate that RAD001 radiosensitizes prostate cancer cells,
specifically PTEN-deficient PC-3 cells. PTEN dephosphorylates the
second messenger PIP3, interrupting PI3K activation of Akt and
decreasing overall flux through the PI3K pathway (26). Mice that are
PTEN heterozygous (+/) have been observed to develop prostatic
intraepithelial neoplasia with nearly 100% frequency, although the
animals may succumb to other cancers before these lesions can
evolve into macroinvasive carcinoma (27, 28). More recently, mice in
which a prostate-specific PTEN deletion has been achieved through
use of prostate-restricted expression of Cre-recombinase have gone
on to develop invasive and metastatic prostate carcinomas with
very high penetrance (29, 30). The relevance of these murine models
is verified by studies of human prostate cancer that have shown that
loss of PTEN is strongly associated with more aggressive cancers
(6, 31). Hence, our data, which show that the PTEN-deficient
prostate cancer cell line PC-3 is particularly susceptible to
radiosensitization via mTOR inhibition, have important clinical
implications.
Despite the considerable evidence supporting the oncogenic
potential of mTOR, the precise mechanism by which mTOR
activation leads to cell transformation has not yet been clearly
defined. Dysregulation of critical components of protein synthesis
machinery may allow aberrant cell cycle signals to facilitate cell
immortality (11). Overexpression of cyclin D1 may play a particularly
crucial role, as recent studies have suggested that this molecule can
restore proliferative potential after rapamycin treatment (32).
Alternatively, mTOR exerts prosurvival influence through hypoxia-
inducible factor 1a, which targets many essential glycolytic enzymes
and protects a variety of solid tumors from their relatively poor
oxygenation status (5, 17, 33). mTOR is also a point of convergence in
balancing the cellular response to growth factors, such as insulin
(12) and multiple stressors, including hypoxia (34) and nutrient
starvation (7). mTOR inhibition with rapamycin has been shown to
induce a starvation-like state in mammalian cells (35). Eukaryotic
cells without a sufficient nutrient source may undergo autophagy as
a self-limited survival strategy, as the breakdown of intracellular
structures provides the organism with a supply of ATP (36).
However, this starvation response will ultimately lead to cell demise
if no rescue can be secured with additional nutrients.
mTOR may be the critical link in mediating PI3K/Akt
prosurvival signals with the suppression of autophagy. It has been
shown that dysfunctional Akt signaling promotes autophagy in
mammalian cells (37), and that the phosphorylation of p70S6
kinase by mTOR (or PDK1) prevents autophagy (38). Although
autophagy has been documented as a form of programmed cell
death enacted in response to various noxious stimuli, including
Figure 4. Inhibiting caspase-dependent
apoptosis shunts radiation-induced cell
death through the autophagic pathway.
A, representative photograph (
1,000) in
PC-3 and DU145 cells showing increased
autophagosome formation in response
to the pan-caspase inhibitor Z-VAD
(10 Amol/L). Quantitative measurement
reveals a >2-fold increase in autophagy in
both PC-3 and DU145 cells treated with
Z-VAD. B, PC-3 cells treated with Z-VAD
show greater radiosensitivity than cells
treated with RAD001; however, the
radiosensitizing effects of both drugs are
additive when combined, yielding the
steepest survival curve in response
to radiation. C, less dramatic
radiosensitization is observed in DU145
cells after treatment with Z-VAD (DER
1.05), but the difference is still significant
(P < 0.006). RAD001 does not significantly
enhance the radiosensitization conferred
by Z-VAD (DER 1.08).
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viruses, toxins, and chemotherapy (36, 39), there is accumulating
evidence that mTOR inactivation also initiates autophagy (19, 40).
These observations lie in accordance with our findings that
suggest mTOR inhibition with RAD001 sensitizes prostate cancer
cells to radiation via the promotion of autophagy.
Our experiments using Z-VAD and Bax/Bak siRNA show that by
blocking caspase-dependent apoptosis, cells can be further
sensitized to the cytotoxic effects of radiation. Similar to RAD001,
Z-VAD also seems to achieve radiosensitization through the
promotion of autophagy. The shunting of cell death through the
autophagic pathway in a caspase-deficient context has been
observed before in MCF-7 breast cancer cells (19). Here, we have
shown that inhibiting apoptosis dramatically induces autophagy
and effectively radiosensitizes prostate cancer cells.
Figure 5. Blocking Bax/Bak–dependent apoptosis also enhances radiation-induced cell killing. A, 20 nmol/L Bax/Bak siRNA blocks production of Bax/Bak proteins.
B, in PC-3 cells, treatment with Bax and Bak antisense RNA (siRNA, 20 nmol/L) produces equivalent radiosensitivity (DER 1.18) to RAD001 (DER 1.18), but
combination treatment results in a synergistic increase in radiosensitivity [DER (versus MS control) 1.26]. C, treating DU145 cells with Bax/Bak siRNA also results
in radiosensitization (DER 1.14). RAD001, although incapable of inducing significant radiosensitization alone, enhances the radiosensitivity obtained with Bax/Bak
siRNA (DER 1.26). D, quantitative analysis of GFP-LC3 transfection shows increased autophagosome formation after radiation and RAD001 independently in both cell
lines, with combination treatment resulting in the highest fraction of transfected cells undergoing autophagy for both PC-3 (0.69 F 0.01) and DU145 (0.48 F 0.06)
cells. E, levels of ATG5 complex and Beclin at 24 hours after being transfected with ATG5 and Beclin siRNA. Note the levels of these proteins in the cells transfected
with control siRNA were set to 1 for comparison. F, treating PC3 or DU145 cells with ATG5/Beclin siRNA results in decreased radiosensitivity.
Radiosensitization of PTEN-Deficient Cancer Cells
www.aacrjournals.org
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Page 6
Establishing that autophagy, in the absence of apoptosis, was
partially responsible for the heightened radiosensitivity conferred
by mTOR inhibition lends further credence to the idea that
the traditional tenets of cancer treatment may exaggerate the role of
p53-dependent cell death. Brown and Wouters (41) have argued that
neither p53 status nor the ability to undergo apoptosis significantly
affects the sensitivity of solid tumor cells to DNA-damaging agents.
For example, many malignant cells have lost the capacity to undergo
normal apoptosis, so it is reasonable to expect alternative modes of
cell death to play a more prominent role in determining the
sensitivity of a particular tumor to genotoxic agents (21). With the
emergence of this new paradigm questioning the apoptotic
sensitivity of solid tumors, our results showing the autophagy
secondary to mTOR and apoptosis inhibition underscore the need
to further investigate how to augment alternative mechanisms of
cell death, such as autophagy, programmed necrosis that can
occur after prolonged or massive autophagy, or mitotic
catastrophe. The genetic constitution of a cancer cell may
determine which mode of death to pursue.
Although this study was conducted in vitro, there is evidence to
suggest that RAD001 efficacy may be improved under in vivo
conditions. For instance, rapamycin is known to impede angio-
genesis (42), and our group has previously shown decreased tumor
vascular density in murine models and sensitized vascular
endothelium when mTOR inhibition is coupled with radiation
(18). This raises the possibility of an additional antitumor effect in
the stroma, and suggests that a greater in vivo response to mTOR
inhibition may be observed than would be predicted by in vitro
studies.
In summary, we believe that this is the first article to show
that mTOR inhibition with RAD001 is capable of radiosensitiz-
ing these cells by amplifying the autophagic pathway of cell death.
This effect is more pronounced in the clinically relevant PTEN-
deficient cell line. As more in vitro studies show the feasibility
and efficacy of mTOR inhibition, methods of monitoring mTOR
levels in vivo will become increasingly important. The duration
of S6K1 inactivation in peripheral blood mononuclear cells cor-
relates with tumor response to mTOR inhibition in rats, and thus
potentially offers a valuable biomarker to assay RAD001 efficacy
in patients (43). Additionally, because mTOR induces GLUT1
expression, preclinical studies have shown the viability of assessing
reduced glucose uptake using
18
FDG-positron emission tomogra-
phy as a way of imaging areas of mTOR inhibition in vivo (44).
These preclinical results provide valuable guidance in designing
clinical trials using mTOR inhibitors.
Acknowledgments
Received 3/2/2006; revised 8/3/2006; accepted 8/10/2006.
Grant support: Vanderbilt Discovery Grant, Vanderbilt Physician Scientist Grant,
Mesothelioma Applied Research Foundation, and Department of Defense grants
PC031161 and BC030542.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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  • Source
    • "Interestingly it has been reported that the termination of autophagy could be mTOR-mediated because the release of the constituents of macromolecules degraded by autophagosomes can in turn reactivate mTORC1, which terminates autophagy [211]. Cao et al. have reported that EVR enhances the cytotoxic effects of radiation on tumor cells (PC-3 and DU145) probably through a drug-related induction of autophagy [212]. "
    [Show abstract] [Hide abstract] ABSTRACT: Sirolimus (SRL) and everolimus (EVR) are mammalian targets of rapamycin inhibitors (mTOR-I) largely employed in renal transplantation and oncology as immunosuppressive/antiproliferative agents. SRL was the first mTOR-I produced by the bacterium Streptomyces hygroscopicus and approved for several medical purposes. EVR, derived from SRL, contains a 2-hydroxy-ethyl chain in the 40th position that makes the drug more hydrophilic than SRL and increases oral bioavailability. Their main mechanism of action is the inhibition of the mTOR complex 1 and the regulation of factors involved in a several crucial cellular functions including: protein synthesis, regulation of angiogenesis, lipid biosynthesis, mitochondrial biogenesis and function, cell cycle, and autophagy. Most of the proteins/enzymes belonging to the aforementioned biological processes are encoded by numerous and tightly regulated genes. However, at the moment, the polygenic influence on SRL/EVR cellular effects is still not completely defined, and its comprehension represents a key challenge for researchers. Therefore, to obtain a complete picture of the cellular network connected to SRL/EVR, we decided to review major evidences available in the literature regarding the genetic influence on mTOR-I biology/pharmacology and to build, for the first time, a useful and specific "SRL/EVR genes-focused pathway", possibly employable as a starting point for future in-depth research projects.
    Full-text · Article · May 2016 · International Journal of Molecular Sciences
  • Source
    • "Existing reports have indicated that combining rapamycin (or its analogs) with radiotherapy results in tumor cell line-dependent radiosensitization. In agreement with previous reports [35][36][37] , in the current study rapamycin-mediated the radiosensitization of only two of the five NSCLC cell lines that were tested. The lack of radiosensitization in the remaining NSCLC cell lines and in the MDA-MB-231 breast cancer cell line was associated with Akt activation following rapamycin treatment. "
    [Show abstract] [Hide abstract] ABSTRACT: Inhibition of mammalian target of rapamycin-complex 1 (mTORC1) induces activation of Akt. Because Akt activity mediates the repair of ionizing radiation-induced DNA double-strand breaks (DNA-DSBs) and consequently the radioresistance of solid tumors, we investigated whether dual targeting of mTORC1 and Akt impairs DNA-DSB repair and induces radiosensitization. Combining mTORC1 inhibitor rapamycin with ionizing radiation in human non-small cell lung cancer (NSCLC) cells (H661, H460, SK-MES-1, HTB-182, A549) and in the breast cancer cell line MDA-MB-231 resulted in radiosensitization of H661 and H460 cells (responders), whereas only a very slight effect was observed in A549 cells, and no effect was observed in SK-MES-1, HTB-182 or MDA-MB-231 cells (non-responders). In responder cells, rapamycin treatment did not activate Akt1 phosphorylation, whereas in non-responders, rapamycin mediated PI3K-dependent Akt activity. Molecular targeting of Akt by Akt inhibitor MK2206 or knockdown of Akt1 led to a rapamycin-induced radiosensitization of non-responder cells. Compared to the single targeting of Akt, the dual targeting of mTORC1 and Akt1 markedly enhanced the frequency of residual DNA-DSBs by inhibiting the non-homologous end joining repair pathway and increased radiation sensitivity. Together, lack of radiosensitization induced by rapamycin was associated with rapamycin-mediated Akt1 activation. Thus, dual targeting of mTORC1 and Akt1 inhibits repair of DNA-DSB leading to radiosensitization of solid tumor cells.
    Full-text · Article · May 2016 · PLoS ONE
  • Source
    • "Thus, mTOR is an attractive therapeutic target, and some mTOR inhibitors have been studied in PCa cells [29]. Preclinical studies suggest that rapamycin analogues CCI-779 and RAD001 inhibit proliferation of human PCa cell lines, especially PTEN-deficient LNCaP and PC-3 cells [25, 32]. In this study, growth curve and colony-formation assays revealed that rapamycin inhibited the growth of LNCaP cells and a lineage-related C4-2 subline [33, 34], and that PC-1 overexpression antagonizes LNCaP cell sensitivity to rapamycin, whereas PC-1-depletion in C4-2 cells had increased rapamycin sensitivity. "
    [Show abstract] [Hide abstract] ABSTRACT: An important strategy for improving advanced PCa treatment is targeted therapies combined with chemotherapy. PC-1, a prostate Leucine Zipper gene (PrLZ), is specifically expressed in prostate tissue as an androgen-induced gene and is up-regulated in advanced PCa. Recent work confirmed that PC-1 expression promotes PCa growth and androgen-independent progression. However, how this occurs and whether this can be used as a biomarker is uncertain. Here, we report that PC-1 overexpression confers PCa cells resistance to rapamycin treatment by antagonizing rapamycin-induced cytostasis and autophagy (rapamycin-sensitivity was observed in PC-1-deficient (shPC-1) C4-2 cells). Analysis of the mTOR pathway in PCa cells with PC-1 overexpressed and depressed revealed that eukaryotic initiation factor 4E-binding protein 1(4E-BP1) was highly regulated by PC-1. Immunohistochemistry assays indicated that 4E-BP1 up-regulation correlates with increased PC-1 expression in human prostate tumors and in PCa cells. Furthermore, PC-1 interacts directly with 4E-BP1 and stabilizes 4E-BP1 protein via inhibition of its ubiquitination and proteasomal degradation. Thus, PC-1 is a novel regulator of 4E-BP1 and our work suggests a potential mechanism through which PC-1 enhances PCa cell survival and malignant progression and increases chemoresistance. Thus, the PC-1-4E-BP1 interaction may represent a therapeutic target for treating advanced PCa.
    Full-text · Article · May 2015 · Oncotarget
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