Download Bevacizumab plus 5-fluorouracil induce growth suppression in the

2149
Bevacizumab plus 5-fluorouracil induce growth
suppression in the CWR-22 and CWR-22R
prostate cancer xenografts
Huynh Hung
Laboratory of Molecular Endocrinology, Division of Cellular
and Molecular Research, National Cancer Centre,
Singapore, Singapore
Abstract
Prostate cancer is the most common malignancy in men.
Although patients with metastatic prostate cancer can
benefit from androgen ablation, most of them will die of
prostate cancer progression to an androgen-refractory
state. In the present study, the effects of docetaxel,
bevacizumab, 5-fluorouracil (5-FU), bevacizumab plus
docetaxel, and bevacizumab plus 5-FU on the growth of
human CWR-22 (androgen-dependent) and CWR-22R
(androgen-independent) prostate carcinoma xenografts
were investigated. We report that i.p. administration of
10 mg/kg docetaxel at 1-week interval, 5 mg/kg/
bevacizumab once every 2 weeks, or 12.5 mg/kg 5-FU,
bevacizumab/docetaxel, or bevacizumab/5-FU weekly to
severe combined immunodeficient mice bearing prostate
cancer xenografts (12 mice per treatment group) for
21 days resulted in 22.5 F 8%, 23 F 7%, 31 F 8%,
22 F 6%, and 81 F 5% growth inhibition, respectively.
Greatest growth suppression was observed in bevacizumab/5-FU treatment. Bevacizumab/5-FU – induced growth
suppression was associated with reduction in microvessel
density, inhibition of cell proliferation; up-regulation of
phosphatase and tensin homologue, p21 Cip1/Waf1 ,
p16INK4a, and p27Kip1; hypophosphorylation of retinoblastoma protein; and inhibition of Akt/mammalian target of
rapamycin pathway. Our data indicate that bevacizumab/
5-FU effectively inhibits angiogenesis and cell cycle
progression and suggest that bevacizumab/5-FU may
represent an alternative treatment for patients with
prostate cancer. [Mol Cancer Ther 2007;6(8):2149 – 57]
Received 1/31/07; revised 6/1/07; accepted 6/29/07.
Grant support: Biomedical Research Council of Singapore grant LS/00/017
(H. Hung).
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.
Requests for reprints: Hung Huynh, Laboratory of Molecular Endocrinology,
Division of Cellular and Molecular Research, National Cancer Centre,
Singapore 169610, Singapore. Phone: 65-436-8347; Fax: 65-226-5694.
E-mail: [email protected]
Copyright C 2007 American Association for Cancer Research.
doi:10.1158/1535-7163.MCT-07-0071
Introduction
Prostate cancer represents the most common disease in men
worldwide and is the second cause of death from malignant
cancers (1, 2). The clinical outcome of prostate cancer is
strongly correlated to its differentiation and tumor grade
(3). Whereas patients with localized disease may be cured
with surgery or radiation, most patients respond initially
to ablation of gonadal androgen production through
either orchiectomy or luteinizing hormone – releasing hormone agonists (4). Eventually, most patients will develop
progressive disease despite continued androgen suppression. The main options currently available for hormonerefractory prostate cancer are second-line hormonal
manipulations, radiation therapy, traditional cytotoxic
chemotherapy, and investigational therapy with novel and
targeted agents (5). Currently, docetaxel plus prednisone is
a standard second-line therapy for hormone-refractory
prostate cancer patients (reviewed in ref. 6).
Prostate cancer arises from multiple genetic and epigenetic aberrations (7). Common aberrations in human
prostate cancer are reduced expression of pRb (8) and
frequent inactivation of phosphatase and tensin homologue
(PTEN; ref. 9). The degree of PTEN deficiency is closely
correlated with activation of the oncogenic kinase Akt (10).
Akt activation affects cell cycle progression through
regulation of cyclin D stability and mRNA translation via
control of phosphorylation of eukaryotic translation initiation factor 4E – binding protein 1 (4E-BP1) and its
dissociation from the mRNA cap binding protein eukaryotic translation initiation factor 4E (11). Targeting of
activated Akt to mouse prostate induces prostate intraepithelial neoplasia, which is reversed following administration of the mammalian target of rapamycin (mTOR)
inhibitor RAD001 (12). It has been reported that the growth
and proliferation of tumors expressing constitutive activation of phosphatidylinositol 3-kinase and Akt or inactivation of PTEN displayed enhanced sensitivity to mTOR
inhibitors (reviewed in ref. 13). Recent studies have found
that mTOR activation is also important for the secretion of
vascular endothelial growth factor (VEGF; ref. 14).
Prostate cancer specimens show increased VEGF expression and vascularity when compared with benign prostatic
hyperplasia and normal prostate tissue (15). An association
does exist between microvessel density in tumor, Gleason
score, metastases, tumor aggressiveness, and progression
(16 – 18). VEGF levels correlate with disease stage and
perhaps survival in the metastatic setting and hormonerefractory prostate cancer (19). In the CWR-22 prostate
cancer xenograft model, the addition of paclitaxel enhances
the antitumor activity of monoclonal VEGF antibody (20).
The safety and efficacy of bevacizumab/docetaxel/
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2150 Bevacizumab/5-FU Inhibits Prostate Cancer Growth
estramustine combination for hormone-refractory prostate
cancer (reviewed in ref. 21) and of bevacizumab/irinotecan/5-fluorouracil (5-FU)/leucovorin combination for
metastatic colorectal cancer (22) have been reported.
Several antiangiogenic agents are under investigation in
clinical trials for prostate cancer, including thalidomide
(23), thalidomide analogues (24, 25), and VEGF receptor
inhibitors such as AZD6474, KRN633, and CEP-7055
(reviewed in ref. 26).
In the present study, we show that addition of bevacizumab to 5-FU – based chemotherapy potently inhibits
tumor growth, angiogenesis, cell cycle progression, and
the Akt/mTOR pathway. This combination holds a
promise as an adjuvant to conventional therapies.
Materials and Methods
Docetaxel (Taxotere) was obtained from Aventis Laboratories. Fluorouracil Injection B.P. was from Mayne Pharma
Plc. Bevacizumab was from Genentech, Inc. Antibodies
against pRb, a-tubulin, cyclin A, cyclin B1, cyclin D1, Cdk4, Cdk-2, p21Cip1/Waf1, p16INK4a, and p27Kip1 were obtained
from Santa Cruz Biotechnology, Inc. Phosphorylationspecific antibodies against mTOR (Ser2448), p70S6 kinase
(Thr421/Ser424), p70S6 kinase (Thr389), S6R (Ser235/236), S6R
(Ser240/242), 4E-BP1 (Ser37/46), 4E-BP1 (Thr70), Akt (Ser308),
Akt (Ser473), pRB (Ser807/811), and pRB (Ser795) and antibodies against 4E-BP1, p70S6 kinase, and mTOR were
obtained from Cell Signaling Technology. Antibodies
against PTEN, CD31/platelet endothelial cell adhesion
molecule-1, and Ki-67 were obtained from NeoMarkers.
Cleaved caspase-3 antibody was from Cell Signaling
Technology. Conjugated secondary antibodies were supplied by Pierce. The chemiluminescent detection system
was supplied by Amersham Pharmacia Biotech.
Tumorigenicity in Severe Combined Immunodeficient
Mice
The study received ethics board approval from the
National Cancer Centre of Singapore and Singapore
General Hospital. All mice were maintained according to
the ‘‘Guide for the Care and Use of Laboratory Animals’’
published by NIH. They were provided with sterilized food
and water ad libitum and housed in negative pressure
isolators with 12-h light/dark cycles.
Prostate cancer xenografts were carried out with male
severe combined immunodeficient (SCID) mice of 9 to
10 weeks of age (Animal Resources Centre, Canning Vale,
West Australia). Androgen-dependent human CWR-22 (27)
and androgen-independent CWR-22R (28) prostate cancer
xenografts were minced under sterile conditions. Approximately 1 10 7 cells were s.c. implanted in both flanks of
male SCID.
To investigate the effects of bevacizumab, docetaxel, and
5-FU on the growth of prostate cancer xenografts, these
drugs were diluted in saline solution at an appropriate
concentration. Mice bearing tumor xenografts were divided
into five groups and each consisted of 12 mice. They were
i.p. injected with 100 AL of saline, 5 mg/kg bevacizumab
every 2 weeks (29), or weekly with 12.5 mg/kg 5-FU (30),
10 mg/kg docetaxel (two doses in total at one-week interval
as described; ref. 31), bevacizumab plus 5-FU, or bevacizumab plus docetaxel for 21 days. Treatments started on
day 7 after tumor implantation. By this time, the tumors
had reached the size of f100 mm3. Tumor growth was
monitored every 2 days by vernier caliper measurement of
the length (a) and width (b) of the tumor. Tumor volume
was calculated as (a b 2)/2. The animals were sacrificed
on day 21 during the treatment because the tumor size in
the controls exceeded 1,500 mm3 in accordance with the
animal care protocol. Body weight and tumor weight were
recorded, and tumors harvested for later analysis.
Efficacy of antitumor agents was determined by %T/C,
where T and C are the median tumor weight (in milligrams) of drug-treated and vehicle-treated mice at day 21
during the treatment, respectively. Ratios of V42% are
considered an active response (Drug Evaluation Branch of
the Division of Cancer Treatment, National Cancer Institute
criteria).
Immunohistochemistry
Five-micrometer sections were dewaxed, rehydrated, and
subjected to antigen retrieval. After blocking endogenous
peroxidase activity and nonspecific staining, the sections
were incubated with antibodies against Ki-67, CD31, and
cleaved caspase-3 (overnight at 4jC). Immunohistochemistry was done using the streptavidin-biotin peroxidase
complex method according to the manufacturer’s instructions (Lab Vision) using 3,3¶-diaminobenzidine as the
chromogen. Sections known to stain positively were
incubated in each batch and negative controls were also
prepared by replacing the primary antibody with preimmune sera. For Ki-67, only nuclear immunoreactivity was
considered positive. The number of labeled cells among at
least 500 cells per region was counted and then expressed
as percentage values. For the quantification of mean vessel
density in sections stained for CD31, 10 random 0.159-mm2
fields at 100 magnification were captured for each tumor
and microvessels were quantified. The data were expressed
as mean F SE.
Western Blot Analysis
To determine changes in indicated proteins, independent
tumors from vehicle-treated (n = 3), bevacizumab-treated
(n = 3 – 4), 5-FU – treated (n = 2 – 3), and bevacizumab/
5-FU – treated (n = 4) mice were homogenized in buffer
containing 20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl,
1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100,
2.5 mmol/L sodium pyrophosphate, 1 mmol/L h-glycerolphosphate, 2 mmol/L Na3VO4, 1 Ag/mL leupeptin, and
1 mmol/L phenylmethylsulfonyl fluoride. One hundred
micrograms of tissue lysate were subjected to Western
blot analysis as previously described (30). Blots were
incubated with indicated primary antibodies and 1:7,500
horseradish peroxidase – conjugated secondary antibodies.
All primary antibodies were used at a final concentration of
1 Ag/mL. The blots were then visualized with a chemiluminescent detection system (Amersham) as described by
the manufacturer.
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Molecular Cancer Therapeutics
Figure 1.
Effects of docetaxel,
bevacizumab, 5-FU, bevacizumab
plus docetaxel, and bevacizumab
plus 5-FU treatments on body weight
at sacrifice, growth rate, and tumor
weight of CWR-22 and CWR-22R
xenografts. The indicated xenografts
were s.c. implanted on the right side
of male SCID mice as described in
Materials and Methods. Mice bearing
hepatocellular carcinoma xenografts
were randomized to one of the four
treatment groups (12 mice per
group) and treated with either vehicle, 10 mg/kg docetaxel, or 5 mg/kg
bevacizumab (BEV ), 12.5 mg/kg
5-FU, bevacizumab plus docetaxel,
or bevacizumab plus 5-FU for 21 d
starting from day 7 after tumor
implantation as described in Materials and Methods. A, body weight at
sacrifice (day 21 during treatment).
B, tumor volume at a given time for
CWR-22R xenografts treated with
various treatments. C, efficacy
(%T/C ) of tested drugs. Ratios of
V42% are considered an active response (Drug Evaluation Branch of
the Division of Cancer Treatment,
National Cancer Institute criteria).
Experiments were repeated at least
thrice with similar results. Columns
with different letter indicate significant differences at P < 0.01.
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2152 Bevacizumab/5-FU Inhibits Prostate Cancer Growth
Statistical Analysis
To obtain the P values, the experiments were repeated
thrice. Differences in tumor growth and expression of
indicated proteins, tumor weight, Ki-67 index, mean vessel
density, and cleaved caspase-3 – positive cells were analyzed by ANOVA.
Results
To study the effects of docetaxel, bevacizumab, 5-FU, and
their combinations on prostate cancer growth, mice
bearing CWR-22 and CWR-22R xenografts were treated
with docetaxel, bevacizumab, 5-FU, bevacizumab/
docetaxel, and bevacizumab/5-FU. Both animal toxicity
and the ability of these treatments to inhibit tumor
formation and progression were determined. In preliminary studies, we found that treatment with a nonspecific
antibody of the same immunoglobulin G isotype had no
effect on tumor growth and was essentially equivalent to
vehicle alone (data not shown). No overt toxicity of
docetaxel, bevacizumab, 5-FU, and the combined therapies
was observed during the course of treatment as defined by
weight loss, unkempt appearance or mortality, and
behavior (Fig. 1A). The efficacy of docetaxel, bevacizumab,
and 5-FU as determined by %T/C is shown in Fig. 1C.
All the treatments significantly inhibited tumor growth
(P < 0.01). Figure 1C shows that T/C ratio in bevacizumab/
5-FU was <0.42 (Drug Evaluation Branch of the Division of
Cancer Treatment, National Cancer Institute criteria),
suggesting that this combination was very active. The
androgen-dependent human CWR-22 was more sensitive
than androgen-independent CWR-22R prostate cancer
(Fig. 1C). Because bevacizumab/5-FU combination exhibits
higher antitumor activity than bevacizumab/docetaxel, this
combination was selected for further studies.
We next examined the association between the antitumor
activity of bevacizumab, 5-FU, and bevacizumab/5-FU
treatments and their ability to inhibit blood vessel
formation in prostate cancer xenografts. Bevacizumab
(Fig. 2C) and bevacizumab/5-FU (Fig. 2D) decreased blood
vessels supplied to the tumors. Mild suppression of 5-FU
on neovascularization was also noticed (Fig. 2B). Table 1
shows the median number of CD31-positive tumor cells
from vehicle-, bevacizumab-, 5-FU – , and bevacizumab/
5-FU – treated tumors on day 21 during the treatment. The
number of microvessels in bevacizumab-treated tumors
was approximately 35% and 17% of that seen in vehicletreated tumors (Table 1). Although bevacizumab/5-FU
potently inhibited angiogenesis, we observed very little
necrosis in bevacizumab/5-FU – treated tumors (data not
shown).
Figure 2. Effects of bevacizumab,
5-FU, and bevacizumab plus 5-FU
therapies on neovascularization of
CWR-22R prostate cancer xenografts. CWR-22R cells were s.c.
implanted on both flanks of male
SCID mice as described in Materials
and Methods. Mice bearing hepatocellular carcinoma xenografts were
randomized to one of the four treatment groups (12 mice per group) and
treated with either vehicle (A), 5-FU
(B), bevacizumab (C), or bevacizumab/5-FU (D) for 21 d starting from
day 7 after tumor implantation as
described in Materials and Methods.
Representative pictures of blood vessel supply to the tumors at sacrifice.
Experiments were repeated thrice
with similar results. Note that bevacizumab/5-FU inhibited the formation of
blood vessels thereby cutting blood
supply to the tumor cells.
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Molecular Cancer Therapeutics
Table 1. Effects of bevacizumab, 5-FU, and bevacizumab plus 5-FU therapies on microvessel density, cell proliferation, and apoptosis of
CWR-22R and CWR-22 prostate cancer xenografts
Xenograft lines
CWR-22R
CWR-22
Treatments
Microvessel density*
Vehicle
Bevacizumab (5 mg/kg)
5-FU (12.5 mg/kg)
Bevacizumab + 5-FU
Vehicle
Bevacizumab (5 mg/kg)
5-FU (12.5 mg/kg)
Bevacizumab + 5-FU
36.5 F 11a
14.5 F 5.4b
26.7 F 9a
6.3 F 4.5c
21.8 F 7.6a
8.4 F 4b
19.1 F 5a
4.2 F 3c
Ki-67 index (%)
36.2
16.3
20.4
7.2
26
11.3
12
5.3
F
F
F
F
F
F
F
F
9a
5.2b
8.2b
4.5c
5a
4.2b
6b
3.4c
Cleaved caspase-3 (%)
9.8
8.1
16.5
5.3
8.4
7.6
14.8
7.1
F
F
F
F
F
F
F
F
4.2a
2.7a
4.9b
3.4a
4a
2.5a
3.9b
2.4a
NOTE: CWR-22R cells were s.c. implanted on both flanks of male SCID mice as described in Materials and Methods. Mice bearing hepatocellular carcinoma
xenografts were randomized to one of the four treatment groups (12 mice per group) and treated with vehicle, 5 mg/kg bevacizumab, 12.5 mg/kg 5-FU, or
bevacizumab plus 5-FU for 21 d starting from day 7 after tumor implantation as described in Materials and Methods. By this time, CWR-22R xenografts had
reached the size of f100 mm3. Mean vessel density, Ki-67 index, and apoptosis in the tumors at harvest (day 21 during treatment) were determined by
immunohistochemical staining with antibodies against CD31, Ki-67, and cleaved caspase-3, respectively. The data are expressed as mean F SE. Differences in
tumor weight, microvessel density, Ki-67 index, and cleaved caspase-3 between vehicle, bevacizumab, 5-FU, and bevacizumab/5-FU groups were analyzed by
ANOVA. Different superscript letter indicates significant difference between values (P < 0.01).
*Mean microvessel density of 10 random 0.159-mm2 fields at 100 magnification.
To examine the antiproliferative and apoptotic effects of
bevacizumab, 5-FU, and combined bevacizumab/5-FU
treatments in vivo, sections were stained with Ki-67 and
cleaved caspase-3 antibodies. Table 1 shows that the Ki-67
labeling index in bevacizumab-, 5-FU – , and bevacizumab/
5-FU – treated tumors was significantly decreased compared with vehicle-treated tumors (P < 0.01). Further
decrease in number of cells stained with Ki-67 antibody
was observed in the combined therapy. The percentage of
cells stained for cleaved caspase-3 was significantly
increased in 5-FU – treated (P < 0.01) but not in bevacizumab- or bevacizumab/5-FU – treated tumors, suggesting
that bevacizumab and bevacizumab/5-FU did not cause
any significant apoptosis (Table 1).
Because PTEN deficiency leads to Akt activation and
triggers initiation of prostate cancer (9), we determined
whether bevacizumab/5-FU – inhitbed prostate cancer
xenograft growth was associated with changes in these
proteins. Figure 3 shows that whereas 5-FU had no effect
on PTEN expression and Akt phosphorylation, bevacizumab slightly increased phosphorylation of Akt at Ser473.
Whereas PTEN expression in bevacizumab/5-FU – treated
samples was increased by 3.6-fold (P < 0.01), phosphorylation of Akt at Thr308 and Ser473 was significantly
decreased (P < 0.01; Fig. 3), suggesting that Akt was
inactivated.
Because phosphatidylinositol 3-kinase/Akt – stimulated
oncogenesis is dependent on mTOR (32), and downstream
targets of mTOR play an important role in regulating cell
cycle progression (33) and angiogenesis (reviewed in
ref. 34), the expression of positive and negative cell cycle
regulators and the abundance of phosphorylation of
mTOR, p70S6 kinase, and 4E-BP1 were investigated.
Figure 3 shows that the levels of phospho-mTOR at
Ser2448, phospho-p70S6 kinase at Thr421/Ser424, and phospho-4E-BP1 at Thr37/46 and Thr70 in tumors derived from
mice treated with bevacizumab/5-FU, but not bevacizumab
or 5-FU, were significantly decreased (P < 0.01). Total
4E-BP1, mTOR, and p70S6 kinase was not altered by any
treatments, suggesting that the mTOR pathway is inactivated. Figure 4 shows that a slight increase in cyclin D1 was
seen in bevacizumab-treated samples. Cyclin B1, cyclin A,
Cdk-2, and Cdk-4 expression was not affected by 5-FU or
bevacizumab treatment. The levels of positive regulators
Cdk-2 and Cdk-4 in bevacizumab/5-FU – treated tumors
were significantly decreased (P < 0.01). Up-regulation of
p21Cip1/Waf1, p16INK4a, and p27Kip1 and inhibition of pRb
phosphorylation at Ser807/811 were seen only in bevacizumab/5-FU – treated tumors. Whereas total pRb was not
significantly altered, hypophosphorylation of pRb at Ser795
was significantly reduced across the treatments. Similar
results were obtained when bevacizumab/5-FU – treated
CWR-22 tumors were analyzed (Supplementary data).1
Discussion
Despite tremendous efforts and resources devoted to
treatment, the incidence and mortality of prostate cancer
have not decreased in the past decades because prostate
cancer cells are barely responsive to chemotherapeutic
agents or radiotherapy (reviewed in ref. 6). Although
patients with metastatic prostate cancer can benefit from
androgen ablation, most of them will die of prostate cancer
progression to an androgen-refractory state. Therefore, an
effective treatment strategy against prostate cancer is
needed to spare the burden of the patients. In the present
study, we show that bevacizumab, docetaxel, and 5-FU,
when given as single agents, inhibit the growth of both
androgen-dependent and androgen-independent prostate
cancer xenografts. The growth of prostate cancer xenografts
is further suppressed when bevacizumab is given with
5-FU but not docetaxel. Bevacizumab/5-FU up-regulates
1
Supplementary material for this article is available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
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PTEN, an upstream down-regulator of phosphatidylinositol 3-kinase/Akt, and inhibits the phosphorylation of Akt,
an upstream positive modulator of mTOR, leading to
inactivation of p70S6 kinase and 4E-BP1. In addition,
bevacizumab/5-FU also enhances the expression of
p21Cip1/Waf1, p16INK4a, and p27Kip1, resulting in hypophosphorylation of retinoblastoma. These events may contribute
to its potent growth inhibition. By inhibiting protein
synthesis, cell cycle progression, and angiogenesis, bevacizumab/5-FU therapy may prove to be useful in maintaining dormancy of micrometastasis and preventing the
development of overt recurrence or metastasis after
surgical resection of a primary tumor.
Both 5-FU and bevacizumab, when administered as
single agents, reduced but did not fully suppress the
growth of CWR-22R and CWR-22 prostate cancer xenografts. However, bevacizumab/5-FU significantly suppressed prostate cancer growth to a greater degree than
single-agent therapy. The mechanism of action of bevacizumab/5-FU is likely to be multifactorial and should be
further investigated using different approaches. Recently,
studies have discovered that the mTOR pathway regulates
tumor angiogenesis as it stimulates VEGF production by
tumor cells (35). It is possible that combined suppression of
both VEGF protein (by bevacizumab) and VEGF expression
(by targeting mTOR via bevacizumab/5-FU) might induce
a synergistic inhibition of prostate tumor angiogenesis.
Thus, one function of bevacizumab/5-FU is to prevent the
tumor mass from expanding by preventing further development of the tumor neovascular network. It has been
Figure 3.
Effects of bevacizumab, 5-FU, and
bevacizumab plus 5-FU therapies on the expression of proteins involved in the PTEN/Akt/
mTOR pathway in CWR-22R xenografts.
CWR-22R cells were implanted and treated as
described in Fig. 2. Lysates from vehicle- and
drug-treated tumors were subjected to Western blot analysis as described in Materials and
Methods. Blots were incubated with the
indicated antibodies. Each lane represents
the tumor from one individual mouse from the
same experiment. Changes in the expression of
the indicated proteins are shown. Experiments
were repeated at least thrice with similar
results.
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Molecular Cancer Therapeutics
Figure 4.
Effects of bevacizumab, 5-FU, and
bevacizumab plus 5-FU therapies on the levels of
positive and negative cell cycle regulators in CWR22R xenografts. CWR-22R cells were implanted
and treated as described in Fig. 2. Lysates from
vehicle- and drug-treated tumors were subjected to
Western blot analysis as described in Materials and
Methods. Blots were incubated with the indicated
antibodies. Each lane represents the tumor from
one individual mouse from the same experiment.
Changes in the expression of the indicated proteins
are shown. Experiments were repeated at least
thrice with similar results.
reported that tumor-associated endothelial cells are targets
of bevacizumab in vivo. These cells express VEGF receptor
and require VEGF for proliferation and survival (36). With
the inhibition of activity by bevacizumab and VEGF
production by bevacizumab/5-FU through inhibition of
mTOR, tumor-associated endothelial cells, whose proliferating frequency is 20 to 2,000 times higher than that of
endothelial cells in normal organs (37, 38), would be more
sensitive to the combined bevacizumab/5-FU treatment.
Supporting this hypothesis, we observe that bevacizumab/
5-FU – treated tumors have a gross reduction in blood
supply (Fig. 2D) and fewer visible blood vessels compared
with bevacizumab and 5-FU.
We have shown that docetaxel, when given at a dose of
10 mg/kg per week for 2 weeks, has moderate activity
against prostate cancer growth. This is in agreement
with the previous study (31). Our present study shows
that bevacizumab, when given together with 5-FU, was far
more effective than monotherapy in treatment of prostate
cancer. Our data, coupled with previous reports (20, 31, 39),
suggest that in prostate cancer, targeting different pathways using both biological and cytotoxic compounds is
needed. The safety and efficacy of bevacizumab/docetaxel/estramustine combination for hormone-refractory
prostate cancer have been reported (reviewed in ref. 21).
In the present study, we observe that docetaxel does not
enhance the antitumor activity of bevacizumab. Experiments are under way to determine why this combination
fails.
Dysregulation of cell cycle control mechanism is an
important carcinogenic mechanism. The p16INK4a/cyclin
D1/pRb pathway is a major regulator of the cell cycle
(reviewed in ref. 40). Whereas bevacizumab/5-FU regulates
the expression of several cell cycle – related components,
including up-regulation of p21Cip1/Waf1, p16INK4a, and
p27Kip1 and inhibition of pRb phosphorylation (P < 0.01;
Fig. 4), such synergistic effects are not observed when 5-FU
or bevacizumab was used as monotherapy, suggesting that
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bevacizumab/5-FU also inhibits cell cycle progression. In
addition, bevacizumab/5-FU may also block cell cycle
progression by inhibiting the Akt activity and downstream
effectors of mTOR, p70S6 kinase, and 4E-BP1. Inactivation
of mTOR pathway may lead to decreased translation of
mRNAs encoding positive regulators of cell cycle progression, such as cyclin D1, and to increased translation of
negative regulators such as p27Kip1, as previously reported
(11, 41).
Our data show that addition of bevacizumab to 5-FU –
based chemotherapy potently inhibits the Akt/mTOR
pathway. This observation has clinical implications because
the growth and proliferation of the tumors with Akt
activation and/or PTEN loss display enhanced sensitivity
to rapamycin and analogues (42). These suggest that
inhibition of Akt/mTOR activity by bevacizumab/5-FU
could significantly contribute to treatment of prostate
cancer. Apart from rapamycin and its analogues, CCI-779
and RAD001, no chemotherapeutic drugs used in treatment
of prostate cancer effectively inhibit mTOR and its
downstream targets. To enhance the antitumor effect of
bevacizumab/5-FU, it is also worth considering to include
mTOR inhibitors, such as CCI-779 and RAD001 (32, 43 – 45),
to bevacizumab/5-FU regimen especially for patients with
nonfunctional PTEN tumors or patients with metastasis
and hormone-refractory tumors. Although our preclinical
data hold much promise, clinical trials are needed to
determine whether bevacizumab/5-FU is as effective as
docetaxel/prednisone (reviewed in ref. 6) or docetaxel/
trastuzumab combination (39) in therapy for metastatic
hormone-refractory prostate cancer.
13. Fingar DC, Blenis J. Target of rapamycin (TOR): an integrator of
nutrient and growth factor signals and coordinator of cell growth and cell
cycle progression. Oncogene 2004;23:3151 – 71.
14. Treins C, Giorgetti-Peraldi S, Murdaca J, Semenza GL, Van
Obberghen E. Insulin stimulates hypoxia-inducible factor 1 through a
phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling
pathway. J Biol Chem 2002;277:27975 – 81.
15. Bigler SA, Deering RE, Brawer MK. Comparison of microscopic vascularity in benign and malignant prostate tissue. Hum Pathol 1993;24:220 – 6.
16. Vesalainen S, Lipponen P, Talja M, Alhava E, Syrjanen K. Tumor
vascularity and basement membrane structure as prognostic factors in T12MO prostatic adenocarcinoma. Anticancer Res 1994;14:709 – 14.
17. Kitadai Y, Haruma K, Tokutomi T, et al. Significance of vessel count
and vascular endothelial growth factor in human esophageal carcinomas.
Clin Cancer Res 1998;4:2195 – 200.
18. Borre M, Nerstrom B, Overgaard J. Association between immunohistochemical expression of vascular endothelial growth factor (VEGF),
VEGF-expressing neuroendocrine-differentiated tumor cells, and outcome
in prostate cancer patients subjected to watchful waiting. Clin Cancer Res
2000;6:1882 – 90.
19. George DJ, Halabi S, Shepard TF, et al. Prognostic significance of
plasma vascular endothelial growth factor levels in patients with hormonerefractory prostate cancer treated on Cancer and Leukemia Group B 9480.
Clin Cancer Res 2001;7:1932 – 6.
20. Fox WD, Higgins B, Maiese KM, et al. Antibody to vascular
endothelial growth factor slows growth of an androgen-independent
xenograft model of prostate cancer. Clin Cancer Res 2002;8:3226 – 31.
21. Armstrong AJ, Carducci MA. New drugs in prostate cancer. Curr Opin
Urol 2006;16:138 – 45.
22. Hurwitz HI, Fehrenbacher L, Hainsworth JD, et al. Bevacizumab in
combination with fluorouracil and leucovorin: an active regimen for firstline metastatic colorectal cancer. J Clin Oncol 2005;23:3502 – 8.
23. Dahut WL, Gulley JL, Arlen PM, et al. Randomized phase II trial of
docetaxel plus thalidomide in androgen-independent prostate cancer.
J Clin Oncol 2004;22:2532 – 9.
24. Capitosti SM, Hansen TP, Brown ML. Thalidomide analogues
demonstrate dual inhibition of both angiogenesis and prostate cancer.
Bioorg Med Chem 2004;12:327 – 36.
References
25. Ng SS, MacPherson GR, Gutschow M, Eger K, Figg WD. Antitumor
effects of thalidomide analogs in human prostate cancer xenografts
implanted in immunodeficient mice. Clin Cancer Res 2004;10:4192 – 7.
1. Denmeade SR, Isaacs JT. A history of prostate cancer treatment. Nat
Rev Cancer 2002;2:389 – 96.
26. Jimenez JA, Kao C, Raikwar S, Gardner TA. Current status of antiangiogenesis therapy for prostate cancer. Urol Oncol 2006;24:260 – 8.
2. Hsing AW, Tsao L, Devesa SS. International trends and patterns of
prostate cancer incidence and mortality. Int J Cancer 2000;85:60 – 7.
27. Wainstein MA, He F, Robinson D, et al. CWR22: androgen-dependent
xenograft model derived from a primary human prostatic carcinoma.
Cancer Res 1994;54:6049 – 52.
3. Gleason DF, Mellinger GT. Prediction of prognosis for prostatic
adenocarcinoma by combined histological grading and clinical staging.
J Urol 1974;111:58 – 64.
4. Goktas S, Crawford ED. Optimal hormonal therapy for advanced
prostatic carcinoma. Semin Oncol 1999;26:162 – 73.
5. Lara PN, Jr., Meyers FJ. Treatment options in androgen-independent
prostate cancer. Cancer Invest 1999;17:137 – 44.
6. Kantoff P. Recent progress in management of advanced prostate
cancer. Oncology (Huntingt) 2005;19:631 – 6.
7. Dawkins HJ, Sellner LN, Turbett GR, et al. Distinction between
intraductal carcinoma of the prostate (IDC-P), high-grade dysplasia (PIN),
and invasive prostatic adenocarcinoma, using molecular markers of cancer
progression. Prostate 2000;44:265 – 70.
8. Brooks JD, Bova GS, Isaacs WB. Allelic loss of the retinoblastoma gene
in primary human prostatic adenocarcinomas. Prostate 1995;26:35 – 9.
9. Porkka KP, Visakorpi T. Molecular mechanisms of prostate cancer. Eur
Urol 2004;45:683 – 91.
10. Trotman LC, Niki M, Dotan ZA, et al. Pten dose dictates cancer
progression in the prostate. PLoS Biol 2003;1:E59.
11. Gera JF, Mellinghoff IK, Shi Y, et al. AKT activity determines
sensitivity to mammalian target of rapamycin (mTOR) inhibitors by
regulating cyclin D1 and c-myc expression. J Biol Chem 2004;279:
2737 – 46.
12. Majumder PK, Febbo PG, Bikoff R, et al. mTOR inhibition reverses
Akt-dependent prostate intraepithelial neoplasia through regulation of
apoptotic and HIF-1-dependent pathways. Nat Med 2004;10:594 – 601.
28. Nagabhushan M, Miller CM, Pretlow TP, et al. CWR22: the first human
prostate cancer xenograft with strongly androgen-dependent and relapsed
strains both in vivo and in soft agar. Cancer Res 1996;56:3042 – 6.
29. Willett CG, Boucher Y, di Tomaso E, et al. Direct evidence that the
VEGF-specific antibody bevacizumab has antivascular effects in human
rectal cancer. Nat Med 2004;10:145 – 7.
30. Huynh H, Soo KC, Chow PK, Panasci L, Tran E. Xenografts of human
hepatocellular carcinoma: a useful model for testing drugs. Clin Cancer Res
2006;12:4306 – 14.
31. Oudard S, Legrier ME, Boye K, et al. Activity of docetaxel with or
without estramustine phosphate versus mitoxantrone in androgen dependent and independent human prostate cancer xenografts. J Urol 2003;
169:1729 – 34.
32. Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT
pathway in human cancer. Nat Rev Cancer 2002;2:489 – 501.
33. Jacinto E, Hall MN. Tor signalling in bugs, brain and brawn. Nat Rev
Mol Cell Biol 2003;4:117 – 26.
34. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes
Dev 2004;18:1926 – 45.
35. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits
primary and metastatic tumor growth by antiangiogenesis: involvement of
vascular endothelial growth factor. Nat Med 2002;8:128 – 35.
36. Tran J, Rak J, Sheehan C, et al. Marked induction of the IAP family
antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial
cells. Biochem Biophys Res Commun 1999;264:781 – 8.
Mol Cancer Ther 2007;6(8). August 2007
Downloaded from mct.aacrjournals.org on May 15, 2017. © 2007 American Association for Cancer Research.
Molecular Cancer Therapeutics
37. Hobson B, Denekamp J. Endothelial proliferation in tumours and
normal tissues: continuous labelling studies. Br J Cancer 1984;49:
405 – 13.
38. Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH, Augustin
HG. Heterogeneity of angiogenesis and blood vessel maturation in human
tumors: implications for antiangiogenic tumor therapies. Cancer Res 2000;
60:1388 – 93.
39. Legrier ME, Oudard S, Judde JG, et al. Potentiation of antitumour
activity of docetaxel by combination with trastuzumab in a human prostate
cancer xenograft model and underlying mechanisms. Br J Cancer 2007;
96:269 – 76.
40. Thomas MB, Abbruzzese JL. Opportunities for targeted therapies in
hepatocellular carcinoma. J Clin Oncol 2005;23:8093 – 108.
41. Fingar DC, Richardson CJ, Tee AR, Cheatham L, Tsou C, Blenis J.
mTOR controls cell cycle progression through its cell growth effectors
S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E. Mol Cell Biol
2004;24:200 – 16.
42. Thimmaiah KN, Easton J, Huang S, et al. Insulin-like growth factor
I-mediated protection from rapamycin-induced apoptosis is independent of
Ras-Erk1-2 and phosphatidylinositol 3¶-kinase-Akt signaling pathways.
Cancer Res 2003;63:364 – 74.
43. Elit L. CCI-779 Wyeth. Curr Opin Investig Drugs 2002;3:1249 – 53.
44. Huang S, Houghton PJ. Targeting mTOR signaling for cancer therapy.
Curr Opin Pharmacol 2003;3:371 – 7.
45. Luo J, Manning BD, Cantley LC. Targeting the PI3K-Akt pathway in
human cancer: rationale and promise. Cancer Cell 2003;4:257 – 62.
Mol Cancer Ther 2007;6(8). August 2007
Downloaded from mct.aacrjournals.org on May 15, 2017. © 2007 American Association for Cancer Research.
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Bevacizumab plus 5-fluorouracil induce growth suppression
in the CWR-22 and CWR-22R prostate cancer xenografts
Huynh Hung
Mol Cancer Ther 2007;6:2149-2157.
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