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Experimental Therapeutics, Preclinical Pharmacology |
Breast Center, Baylor College of Medicine, Houston, Texas 77030 [K. W., H. T. K., S. G. H., S. K. M., P. H. B.]; Department of Medicine, Division of Medical Oncology [J. L. R., D. M.], and Department of Pharmacology [J. G. K.], The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284; Department of Retinoid Research, Ligand Pharmaceuticals, Inc., San Diego, California 92121 [W. W. L., M. M. G., M. A. S.]; and Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892 [J. E. G.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.002). Histological analysis of the mammary
glands from vehicle and treated mice demonstrated that 9cRA treatment
also did not affect normal mammary gland development.
Immunohistochemical staining of normal and malignant breast tissue and
Western blot analysis demonstrated that SV40 Tag expression was not
affected by treatment with retinoids. Single doses of 10 and 50 mg/kg
resulted in peak plasma concentrations of 3.4 and 6.71
µM, respectively. Daily doses of 9cRA for 28 days
resulted in plasma concentrations of 0.86 and 1.68 µM,
respectively, concentrations consistent with that seen in humans
treated with 9cRA in clinical trials. These results demonstrate that
9cRA suppresses mammary carcinogenesis in transgenic mice without any
major toxicity and suggest that retinoids are promising agents for the
prevention of human breast cancer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50%, there
remains a need to reduce breast cancer incidence further. Therefore, we
have investigated the activity of other promising chemopreventive
agents to suppress breast carcinogenesis in preclinical models. Some of
the most promising chemopreventive agents include retinoids, vitamin D
analogues, dehydroepiandrosterone derivatives, monoterpenes, and
difluoromethylornithine. Of these, retinoids have previously
been shown to suppress cancer development in animals and humans.
Retinoids are vitamin A analogues that play an important role in
regulating cell growth, differentiation, and apoptosis (3
, 4) . These hormones function by binding to specific
RARs3
(RAR 
,
ß, and 
) and specific RXRs (RXR , ß, and )
(5)
. Retinoid receptors are nuclear DNA-binding proteins
that regulate gene expression by binding to retinoic acid-responsive
elements in the promoters of target genes. The RAR and RXR proteins are
expressed differently during development and differentiation
(6)
, and various isoforms of these proteins can
heterodimerize to produce a variety of complexes to regulate different
sets of retinoid-induced genes.
In vitro studies have shown that retinoids can inhibit the growth and invasion of cancer cells and induce apoptosis. Several laboratories, including ours, have found many different cell lines to be significantly growth inhibited by retinoids (7, 8, 9, 10) . The retinoid used in the present study, 9cRA, has been found to inhibit proliferation, induce differentiation, and induce apoptosis in a variety of cell lines including the breast cancer cell line MCF-7 and the human leukemia cell line HL-60 at micromolar concentrations (9, 10, 11, 12, 13, 14, 15) . This retinoid binds both RAR and RXR receptors and thus is able to activate both RAR- and RXR-dependent pathways.
In humans, retinoids have been used in clinical trials for the treatment of leukemia (16 , 17) and squamous cell carcinomas of the skin and cervix (18, 19, 20) . Retinoids have also been used in the prevention of cervical, head and neck, and lung cancers (21, 22, 23) . All-trans-retinoic acid was effective against the development of primary cervical cancers (24) , whereas isoretinoin (13-cis-retinoic acid) was effective against second primary tumors of head and neck squamous cell carcinoma (25) . Retinyl palmitate significantly delayed development of second primaries of lung cancer (26) , and polyprenoic acid was shown to prevent second primary tumors of the liver (27) . These studies suggest that retinoids may be useful for the prevention of breast cancer.
Several in vivo models exist for the study of breast cancer, including chemically induced mammary tumor models in rats using 7,12-dimethylbenz(a)anthracene (DMBA) and NMU to induce mammary tumors, as well as several transgenic mouse models (28, 29, 30, 31) . The ability of retinoids to prevent mammary tumorigenesis has been extensively investigated with the chemical carcinogenesis models. Moon et al. (32, 33, 34) found that retinyl acetate and N-(4-hydroxyphenyl)-retinamide (fenretinide) reduced tumor incidence and multiplicity and also increased the latency of 7,12-dimethylbenz(a)anthracene or NMU-induced mammary cancers. Similar results were found in the NMU-induced model using an RXR-selective retinoid, LGD1069 (35 , 36) . 9cRA has also been shown to inhibit mammary carcinogenesis induced by NMU in Sprague Dawley rats (37) . Although previous studies have shown that retinoids suppress mammary carcinogenesis in chemically treated rats, the relevance of these carcinogen-induced models to human breast cancer has been questioned. The tumors that arise in these animals contain mutations in ras genes (28) , which are a rather uncommon event in human breast cancer (38) .
We have investigated the ability of 9cRA to inhibit mammary tumorigenesis in a transgenic mouse model, the C3(1) -SV40 Tag mouse model. These mice express the SV40 Tag in breast tissue and develop tumors that histologically are similar to high grade human breast cancer (39) . In addition, the SV40 Tag causes inactivation of the p53 and Rb genes, tumor suppressor genes that are commonly mutated in human breast cancer (40 , 41) . Another attractive feature of the SV40 Tag transgenic mouse model is that the tumors that develop arise through a multistep process, similar to that occurring in humans. Thus, female transgenic mice develop mammary hyperplasia by 3 months of age, which is followed by the development of carcinoma in situ lesions, which in turn is followed by invasive adenocarcinoma (reaching 100% incidence by 6 months). In addition, some of the mice develop metastases to the lungs. All these features make this an attractive model to investigate the efficacy of chemopreventive agents. Using these SV40 Tag transgenic mice, we investigated whether 9cRA would affect mammary tumor development in this model. Our data demonstrate that 9cRA suppresses mammary tumorigenesis in these transgenic mice and suggest that 9cRA may be useful for the prevention of breast cancer.
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MATERIALS AND METHODS |
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Treatment and Data Collection.
9cRA (Panretin, LGD1057, Mr 300;
Ligand Pharmaceuticals, Inc., San Diego, CA) was suspended in purified
sesame oil (Croda, Inc., Mill Hall, PA). 9cRA was administered p.o. to
animals by a 20-gauge gavage needle in a volume of 0.1 ml containing
either 10 or 50 mg/kg 9cRA. The animals were treated daily with either
sesame oil (vehicle) or 10 or 50 mg/kg 9cRA from an age of 5 weeks
until the age of 6 months. Tumor measurements were made biweekly with
electronic calipers (Mitutoyo, Utsonomiya, Japan), and tumor volume was
determined by multiplying the square of the width by the length and
dividing by 2. Individual tumor size and tumor location for each animal
were recorded. Weights of all mice were recorded monthly.
At the time of sacrifice, each tumor was resected and separate portions were (a) processed for histological analysis, (b) used to isolate protein for Western blot analysis, and (c) explanted into tissue culture to prepare in vitro tumor cell lines. These cell lines were grown in DMEM containing 10% FBS, 1% glutamine, 1% penicillin/streptomycin, and 1% Fungizone.
Histological Analysis.
Tumor samples were fixed in 10% neutral buffered formalin (10%
formaldehyde, phosphate-buffered) overnight and then embedded in
paraffin. Tissue sections were then mounted on slides and processed for
either H&E staining or immunohistochemical staining. H&E staining was
conducted by cutting 4-µm tissue sections and deparaffinizing in
xylene. Sections were rehydrated in ethanol and running water and then
incubated with hematoxylin for 7 min. Samples were destained in running
water and then fixed in acidified alcohol and ammonia. Slides were then
incubated in eosin for 2 min, rinsed in alcohol and xylene, and mounted
for evaluation.
For immunohistochemical studies, tissue sections were cut at 4 µm and mounted onto slides. Slides were deparaffinized, and then endogenous peroxidase was blocked with 0.1% sodium azide in 3% hydrogen peroxide in 1 x auto buffer. Slides were then rinsed in PBS, and nonspecific binding was blocked with 10% albumin. SV40 Tag was stained using mouse anti-SV40 large Tag monoclonal antibody (1:50; PharMingen, San Diego, CA) followed by a biotinylated rabbit antimouse IgG antibody (1:100). The slides were then incubated with streptavidin-horseradish peroxidase at 1:100, and peroxidase activity was visualized using DAB chromagen intensified with 0.2% osmium tetroxide. Counterstaining was done with 1% methyl green.
Western Blot Analysis of Tumors.
Analysis of tumor powders was done by grinding the tumor and suspending
in 5% SDS. Samples were heated for 5 min at 95°C and spun in a
microfuge to pellet debris. Protein extracts in the supernatant were
run on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose
membrane (Hybond ECL; Amersham Life Sciences, Arlington Heights, IL).
Membranes were probed with mouse anti-SV40 large Tag monoclonal
antibody and then with antimouse IgG conjugated to horseradish
peroxidase (Amersham Life Sciences). SV40 Tag protein was visualized
using ECL (Amersham Life Sciences).
Cell lines derived from tumor explants were treated with either DMSO (vehicle) or 9cRA for 0, 6, 24, and 48 h. Cells were pelleted and lysed in 50 mM Tris (pH 6.8), 2% SDS. Protein extracts were run on a 10% SDS-polyacrylamide gel and analyzed as before.
Plasma Concentration of Retinoids.
After blood was collected in a heparinized tube, the plasma was
separated by centrifugation and transferred into an opaque tube.
Samples were stored under nitrogen gas at -70°C until ready for
analysis. Plasma from three or four mice at each time point was pooled
to provide a sufficient volume for analysis of 9cRA concentration. The
plasma was extracted with 5 volumes of cold methanol, centrifuged, and
dried in a vacuum. Residues were reconstituted in mobile
phase (70% acetonitrile/30% 10 mM ammonium
acetate, adjusted to pH 3 with glacial acetic acid) and injected into a
Zorbax SB C18 column (5 µm, 4.6 x 250 mm,
MAC-MOD Analytical, Inc., Chaddsford, PA) at 40°C using a 1-ml/min
flow rate with UV detection at 330 nm. Standards were prepared by
spiking control plasma and were analyzed as above to generate a
standard curve. Peak areas of the samples were quantified using linear
regression (weighted 1/X) of the standard curve. The area
under the plasma time curve from 0 to the last time point was
determined by linear trapezoidal approximation.
Statistical Analysis of Results.
Two outcome measures, tumor-free survival and tumor multiplicity, were
considered in this study. Tumor-free survival was defined from time of
birth to first appearance of a tumor. Tumors were defined as palpable
masses 
100 mm3
. Tumor-free survival curves were
estimated by the Kaplan-Meier product limit method and compared by the
generalized Wilcoxon test. Similar analysis was performed using a log
rank test that yielded smaller P values than the generalized
Wilcoxon test for each curve. However, to avoid confusion, we report
P values from the more conservative generalized Wilcoxon
test. (In our experience, the generalized Wilcoxon test is the most
appropriate test for these kinds of experiments. The log rank test is
appropriate when differences between tumor-free survival curves can be
described by proportional hazards. For nonproportional hazard
differences between survival curves, such as those in our experiments,
the generalized Wilcoxon test is most appropriate.) We also evaluated
tumor multiplicity by counting the total number of tumors occurring in
each animal up to the time of sacrifice. Tumor multiplicity was
summarized by means and standard errors, and compared by Wilcoxon rank
sum tests. Tumor incidence at a particular time is often also used as
an end point in chemoprevention studies. Because tumors develop in
these animals over a wide time period, we instead have reported
tumor-free survival as a function of time. Incidence at any time point
can be obtained from these curves by subtracting the proportion free of
tumors at any given time from 1.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, which shows dysplastic lobules
within the ducts of normal mammary glands, ducts swollen with
transformed cells (carcinoma in situ lesions), and poorly
differentiated carcinomas which invade the surrounding stroma.
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Fig. 2
shows a plot of the proportion of
animals free of tumor versus age in days. Mice treated with
vehicle show a median time to tumor development of only 140 days. Tumor
development began at 110 days, and by 180 days (6 months) all vehicle
mice had developed tumors. The time to tumor development curve of the
mice treated with low dose 9cRA (10 mg/kg) showed no significant
difference to the curve of the control mice. However, the mice treated
with high dose 9cRA (50 mg/kg) showed a delay in the time to tumor
development with the median time to tumor development delayed to 167
days.
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shows median time to tumor
development, tumor incidence, and number of tumors per mouse (tumor
multiplicity) in mice treated with vehicle, low dose (10 mg/kg), or
high dose (50 mg/kg) of 9cRA. Similar median time to tumor development
was seen in the control mice and the mice treated with 10 mg/kg of 9cRA
(140 days). However, mice treated with the 50-mg/kg dose of 9cRA
showed a significant delay in time to tumor development (167 days,
P = 0.05 by the generalized Wilcoxon test). At the
completion of the experiment, 100% of the mice treated with vehicle or
low dose of 9cRA developed tumors; whereas 70% of mice treated with
high dose of 9cRA developed tumors. Thus, 30% of the high dose
9cRA-treated mice failed to develop tumors by 6 months.
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0.002 as
assessed by ANOVA). There was no significant difference in the average weight of the mice treated with either the vehicle or 9cRA from day 1 to 130 (after 130 days, the weights diverged in animals with large tumors; data not shown). These results suggest that the cancer-suppressive effect of 9cRA is not due to general weight loss. Cutaneous toxicities from the retinoids were observed in mice treated with 50 mg/kg 9cRA, including erythema of their ears (in 100% of the high dose-treated animals but not in the control and low dose-treated animals).
9cRA Does Not Affect Expression of the SV40 Tag Transgene.
To determine whether the tumor-suppressive effects of 9cRA
results were due to down-regulation of the expression of the SV40
transgene, several analyses were conducted. Western blot analysis to
assess SV40 Tag protein levels was done as described above. The results
are shown in Fig. 3A
and
demonstrate that tumors from both control and 9cRA-treated mice express
similar levels of the transgene. These results were further
supplemented by immunohistochemistry to detect SV40 Tag expression in
normal and malignant mammary tissue from these mice. As shown in Fig. 3
B, the transgene is expressed in the normal ducts of both
groups of mice. In addition, tumors from both groups also express
similar amounts of the SV40 Tag.
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C, levels of SV40 Tag in these
tumor explants do not change with 9cRA treatment.
Histological analysis was also conducted using mammary tissue samples
from control and 9cRA-treated mice to determine whether 9cRA affects
the morphology of normal or malignant mammary glands in these mice. A
representative example of the tumors from vehicle- and 9cRA-treated
mice is shown in Fig. 4
. Comparison of
all tumor samples from vehicle- and retinoid-treated mice showed no
significant difference in morphology or nuclear grade. These results
suggest that 9cRA treatment does not induce differentiation of the
tumor cells in these mice. However, a slight reduction in mitotic index
was observed in the retinoid-treated animals as compared with the
control mice (data not shown).
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Fig. 5A
shows the average
plasma concentration over time after a single oral dose of 9cRA (either
10 or 50 mg/kg). As shown in Table 2
, the
Tmax for the 10-mg/kg dose was 60 min
versus 30 min for the 50-mg/kg dose. Levels of 9cRA were
undetectable after 6 h of time. Peak plasma concentrations
achieved after a single dose were 3.40 and 6.71
mM for 10 and 50 mg/kg, respectively, correspond
with concentrations found to inhibit growth in vitro.
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shows the average plasma concentration over
time after a dose of 9cRA in mice that were treated daily with either a
10-mg/kg (low dose) or a 50-mg/kg (high dose) of 9cRA for 28 days. As
shown in Table 2
, the maximum levels of retinoid concentration in the
blood were at 30 min for both the low and high doses. Plasma
concentrations of 9cRA after a single dose in these chronically treated
mice were lower at maximal levels (0.86 and 1.68
mM for 10 and 50 mg/kg, respectively). Area under
the curve values on day 28 were 86 and 78% lower than the day 1 values
at 10- and 50-mg/kg dose levels respectively. These results suggest
that chronic dosage of 9cRA induces enzymes that metabolize 9cRA. The results from the pharmacokinetic studies show that the dose of 50 mg/kg used in these studies can achieve peak plasma levels of up to 6 µM, after a single dose, with levels of 1–2 µM in chronically treated animals. These plasma levels are consistent with levels achieved in humans in recent Phase I clinical trials of 9cRA (42 , 43) . Such plasma levels represent total 9cRA, much of which is bound to plasma proteins. The biological effects of 9cRA are likely induced by intracellular 9cRA that binds to retinoid receptors. The concentration of intracellular 9cRA in the mammary epithelial cells is likely to be much lower than our reported plasma levels. However, to compare the present studies with those in humans, we have monitored total 9cRA levels.
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DISCUSSION |
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The suppression of the tumor development by 9cRA in the SV40 Tag mice is modest compared with the dramatic suppression by 9cRA previously reported by Anzano et al. (37) in NMU-treated rats. This difference in efficacy may occur because the tumors arise by different pathways in the two models. In the NMU rat model, the NMU carcinogen induces ras mutations that promote tumor formation. ras mutations activate the MAPK pathway that may be blocked by retinoids. Indeed retinoids are known to inhibit AP-1 transcription factors downstream of the ras-MAPK pathway (44, 45, 46) . The tumors that arise in the NMU-treated rats are also ER positive, and the antiestrogen tamoxifen dramatically suppresses tumorigenesis in this model (37 , 47) . Thus, activation of the MAPK pathway and the dependence on estrogen signaling may cause NMU-induced tumorigenesis to be particularly sensitive to retinoids.
Our data suggest that SV40 Tag-induced tumorigenesis is also sensitive to retinoids, but less so than NMU-induced rat tumors. Tumorigenesis in SV40 Tag mice is caused by inactivation of the p53 and Rb tumor suppressor genes. The present results indicate that cells transformed by p53 and Rb inactivation are less sensitive to retinoids than are cells transformed by ras activation. Recent studies by us also suggest that tumors arising in the SV40 Tag mice are less dependent on estrogen. Antiestrogens fail to prevent tumor formation in the SV40 Tag mice, and the mammary tumors that arise in these mice are ER negative.4 Thus, the C3(1) SV40 Tag mice represent a model of ER-negative breast tumorigenesis. Such ER-negative cells may be less sensitive to retinoids. Our data, showing modest suppression of mammary tumorigenesis by 9cRA, along with those of Anzano et al. (37) showing more dramatic suppression in NMU-induced rats, suggest that retinoids can suppress the development of both ER-positive and ER-negative tumors but that retinoids may more effectively suppress ER-positive, estrogen-dependent tumors.
The C3(1) -SV40 Tag model, in which SV40 Tag inactivates p53 and Rb, is a model of human breast tumors that arise by p53 or Rb inactivation. p53 and Rb mutations are commonly seen in human breast cancers (39 , 48) . Inactivation of p53 by SV40 Tag serves as a model of tumors that lose p53 function, either through loss of p53 expression or through the expression of a mutant p53 that functions as a dominant negative mutant. However, some human breast cancers have gain-of-function p53 mutations (49) . Such mutations likely cause cancer through additional molecular mechanisms involving transcriptional regulation and induction of genomic instability (50) . The model used in this study is not an accurate model for breast tumors arising as a result of such gain-of-function p53 mutants. However, the C3(1) SV40 Tag mice represent a model of loss-of-function p53 mutations and thus represent an advance in modeling human breast cancer. The present results suggest that retinoids will suppress the development of breast cancers that arise through p53 mutation, at least those that have dominant negative or loss-of-function mutations.
Although retinoids have been shown to induce differentiation in mammary cells (51 , 52) , we did not observe a shift in the morphology of the mammary epithelial cells from 9cRA-treated mice as compared with vehicle-treated mice. The most likely effect of 9cRA is therefore an antiproliferative effect, leading ultimately to a delay in tumor development. Studies by Crist et al. (53) suggest that retinoids may exert their effects on the earlier stages of tumorigenesis. We measured the incidence of dysplasia and carcinoma in situ in tumors arising in vehicle- or retinoid-treated mice and found no difference between the two groups in the incidence of these early lesions.5 Thus, their preliminary results suggest that retinoids are exerting their effects at all stages of tumor development. However, all of these premalignant lesions were observed in the context of invasive cancers that had developed in these mice. To definitively determine whether retinoids suppress the development of premalignant lesions, we will need to assess the effect of retinoids at earlier time points, before invasive tumors develop.
In the present study, mice were treated with 9cRA at an early age to prevent tumor development. This was necessary due to the very rapid tumorigenesis in the SV40 Tag mice. However, if retinoids are to be used in women for breast cancer prevention, it is likely that they will be first used in selected groups only. Because many retinoids are teratogenic, the presently available retinoids will likely be initially limited to postmenopausal women. To make retinoids more generally useful, drug development efforts are now focused on developing less toxic, nonteratogenic retinoids.
The ability of retinoids to suppress tumor development in this and other models of mammary tumorigenesis suggests that 9cRA or possibly other less toxic retinoids are promising agents for the prevention of breast cancer. Retinoids may be most useful when combined with antiestrogens to effectively prevent breast cancer development. Studies have already been conducted examining the effects of retinoids in combination with tamoxifen (37) and with other newer generation-selective ER modulators (54) . Through continued studies of synthetic retinoids, it will be possible to identify retinoids capable of suppressing tumorigenesis while avoiding the toxicity of naturally occurring retinoids. Further research into the mechanisms by which retinoids suppress tumorigenesis will help to clarify the role of retinoids in chemoprevention of breast cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH Grant SR01CA78480
(P. H. B.), a P30CA54174 Cancer Center Support Grant to the San
Antonio Cancer Institute, The V Foundation (K. W.), and NIH Grant CA
54174 (J. G. K.). 
2 To whom requests for reprints should be
addressed, at Breast Center, Baylor College of Medicine, One Baylor
Plaza, MS 600, Houston, TX 77030. Phone: (713) 798-1600; Fax: (713)
798-1642; E-mail: pbrown{at}bcm.tmc.edu
3 The abbreviations used are: 9cRA,
9-cis-retinoic acid; RAR, retinoic acid receptor; RXR,
retinoid X receptor; Tag, T antigen; ER, estrogen receptor; DMBA,
7,12-dimethylbenz[a]anthracene; NMU,
N-nitroso-N-methylurea; MAPK,
mitogen-activated protein kinase.
4 K. Wu, J. E. Green, S. K. Mohsin, and P. H.
Brown, unpublished observations.
5 K. Wu, S. K. Mohsin, and P. H. Brown,
unpublished observations.
Received 9/30/99; revised 5/22/00; accepted 6/12/00.
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