Comparative effects of lovastatin on mammary and prostate oncogenesis in transgenic mouse models

Masa-Aki Shibata1,2, Claudine Kavanaugh2, Eiko Shibata2, Hideaki Abe1, PhuongMai Nguyen3, Yoshinori Otsuki1, Jane B. Trepel3 and Jeffrey E. Green2,4

1 Department of Anatomy and Biology, Osaka Medical College, 2–7, Daigaku-machi, Takatsuki, Osaka 569-8686, Japan,
2 Laboratory of Cell Regulation and Carcinogenesis, Division of Basic Science, Building 41, Room C619, 41 Library Dr. and
3 Medicine Branch, National Cancer Insititute, National Institutes of Health, Bethesda, MD 20892, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effects of lovastatin, a potent inhibitor of HMG CoA reductase, on experimental mammary and prostate oncogenesis, were studied in vitro and in vivo. Lovastatin inhibited cell growth in vitro in a dose-dependent manner for both mammary and prostate cancer cell lines, which was associated with p53-independent apoptosis. Flow cytometric analyses of lovastatin-treated mammary and prostate cancer cells demonstrated cell-cycle G1 arrest, as well as decreases in S and G2/M fractions. p21Waf1 and p27Kip1 were induced by lovastatin in both types of cancer cells. Gene expression profiling of cells treated with lovastatin, however, was remarkable for a paucity of transcriptional changes induced by lovastatin. Treatment with lovastatin for 4 weeks did inhibit the formation of pre-neoplastic mammary intraepithelial neoplasias (MIN) in vivo, but not invasive carcinomas in the C3(1)/SV40 TAg transgenic model of mammary cancer. The decreased multiplicity of MIN lesions was associated with increased levels of apoptosis in these lesions. However, cell proliferation in the mammary lesions was not significantly different between lovastatin-treated and control mice 1 day after lovastatin treatment. In female mice treated with lovastatin for 12 weeks, there was a tendency for reduced tumor volume, which did not reach statistical significance. However, lovastatin did not suppress any lesion formation in the prostate of C3(1)/SV40 TAg transgenic male mice. Our results suggest that as lovastatin exerts an inhibitory effect on the development of early mammary lesions of mammary carcinogenesis, this compound may be useful for the chemoprevention of mammary cancer and might have utility as an adjuvant in breast cancer therapy. The chemopreventive effects of lovastatin in vivo, however, may be tissue-specific.

Abbreviations: HMG-CoA, hydroxymethylglutaryl-coenzyme A; MIN, mammary intraepithelial neoplasias; PIN, prostatic intraneoplasia


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prostate and mammary cancers are the most common malignant diseases in the Western world and have become the second leading cause of cancer deaths in men and women in the US (1). The prevalence of both cancers has continued to rise significantly during the past several decades. According to recent estimates (1), approximately 317 000 men will be diagnosed with prostate cancer and 41 000 will die of the diseases in the US. For women, the cumulative lifetime risk of developing breast cancer is 12%, whereas the lifetime mortality risk has been estimated to be 3.5% (2). Finding potent but safe chemotherapeutic agents for these cancers will have tremendous impact on public health.

Lovastatin is a competitive inhibitor of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis, and has been extensively used for the treatment of hypercholesterolemia (3). Generally regarded as a safe agent, other properties of lovastatin have been identified which suggest that it may have chemotherapeutic effects. Lovastatin leads to the arrest of the cell cycle at the G1 phase (4) and induces two cell-cycle inhibitors, p21Waf1 or p27Kip1, in breast cancer cells (5). Apoptosis has been induced in prostate cancer cells (6) and associated stromal cells (7) by lovastatin.

We have described previously the development of transgenic mice carrying the C3(1)/SV40 TAg construct in which the 5' flanking region of the C3(1) component of the rat prostate steroid binding protein drives the expression of the SV40 early region producing both large T (TAg) and small-t antigens in the epithelium of both the mammary and prostate glands. Mammary and prostate cancers arise in these mice over a predictable time course (8) and histologically resemble the human diseases (911).

Previous in vivo studies have demonstrated that lovastatin inhibits tumor growth and metastasis of implanted mouse mammary tumor cells (12), lung tumor formation of A/J mice initiated with a lung carcinogen (13) and metastasis of transplanted mouse melanoma cells (14). No studies, however, have examined whether lovastatin can suppress the development of spontaneously arising mammary and prostate tumors in a transgenic model system.

In this study we have determined whether lovastatin has antiproliferative effects on mammary and prostate cell lines derived from C3(1)/TAg transgenic tumors (15) and whether these effects could be correlated to effects of lovastatin on the spontaneous development and progression of mammary and prostate tumors in the C3(1)/TAg transgenic mice. Our results indicate that while lovastatin had significant suppressive effects on proliferation of the mammary and prostate cells in vitro, its administration in vivo only led to a diminution of pre-invasive mammary intraepithelial neoplasia (MIN) lesion formation associated with increased apoptosis, but not to reduced carcinoma formation. This may indicate that lovastatin was given relatively late in the course of lesion development or that the early mammary lesions overcome the inhibitory effects of lovastatin. No inhibition of prostate lesion development was observed at any stage, suggesting that the effect of lovastatin may be tissue-specific. Gene expression profiling using cDNA microarrays did not identify major lovastatin-induced changes at the transcriptional level.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental compound
Lovastatin was obtained from Merck & Co. (Rahway, NJ) through the generosity of Dr A.W.Alberts. The inactive lactone form of lovastatin was converted to the active dihydroxy-open acid form as described previously (4).

Animals and mammary carcinoma cell lines
C3(1)/TAg transgenic mice have been described previously (810) and were maintained by breeding with wild-type FVB/N mice. Transgenic animals were identified by slot blot utilizing a 32P-labeled SV40-specific probe hybridized to mouse tail DNA. All manipulations of mice were performed in accordance with the guidelines of the Animal Care and Use Committee and in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, 1985), under an animal study proposal (NCI-FCRDC-98–003) approved by the NIH Animal Care and Use Committee. Animals were killed by CO2 asphyxiation.

The mammary carcinoma cell line M6 was established from female C3(1)/TAg transgenic mice (C.L.Jorcyk et al., submitted for publication) and the prostate carcinoma cell line Pr14 was derived as described previously (15). Both cell lines were grown in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum with 5% CO2.

In vitro studies
Cell proliferation.
M6 and Pr14 cells (1x104 cells) were plated in 6-well plates the day before lovastatin treatment. A dose–response curve to lovastatin was determined for each of the carcinoma cells studied. Cells were incubated with various concentrations of lovastatin (0–40 µM) for 48 h, then stained with trypan blue and counted using a hemocytometer. In addition, the response to lovastatin over time was determined by treating cells with or without 10 µM lovastatin for 24, 48 and 72 h. To identify the apoptotic and/or dead cells within a culture plate, cells were stained with 27 µM acridine orange and 25 µM ethidium bromide and then examined using fluorescent microscopy.

DNA laddering.
M6 cells grown to 70% confluency in 150 cm2 flasks and treated with or without 10 µM lovastatin for various time periods, immediately rinsed with Dulbecco’s phosphate-buffered saline, scraped and harvested. To extract low molecular weight DNA, cell pellets were incubated with lysis buffer (10 mM Tris–HCl, pH 7.4/10 mM EDTA, pH 8.0/0.5% Triton X-100) on ice for 10 min, centrifuged and the supernatants were treated with 400 µg/ml RNase A at 37°C for 1 h, and with 400 µg/ml proteinase K at 37°C for 1 h, followed by precipitation with isopropyl alcohol. To detect internucleosomal DNA fragmentation, 1 µg of DNA was 32P-labeled with 2.5 U of Klenow fragment of DNA PolI in the presence of 0.5 µCi of [{alpha}-32P]dCTP for 30 min at room temperature. The labeled DNA was fractionated on 1.5% modified agarose gels (TreviGel 500: Trevigen, Gaithersburg, MD). Gels were then dried and directly exposed to X-ray film at room temperature for 1 full day.

Northern blot.
A SacII–KpnI fragment of the mouse p53 DNA (kindly provided by Dr Tyler Jacks, Massachusetts Institute of Technology, Cambridge, MA) was labeled with [{alpha}-32P]dCTP by random oligonucleotide-primed synthesis. Five µg of total RNA was electrophoresed on a 1.0% formaldehyde agarose gel, transferred to a nylon membrane and fixed by UV cross-linking. Membranes were hybridized with 32P-labeled probes and washed using standard protocols. The membranes were then exposed to X-ray film at -70°C for varying periods of time.

Flow cytometry for cell-cycle distribution.
M6 and Pr14 cells were harvested after 24 h of treatment with 10 µM lovastatin and flow cytometric analysis was conducted. The measurements were performed on cell suspensions following the trypsinization of cells and their fixation in cold 70% ethanol. The cells were treated with 100 µg/ml RNase A for 30 min at 37°C, rinsed with PBS, stained with 5 µg/ml propidium iodide for 30 min on ice and then analyzed with a flow cytometer (EPICS Elite ESP; Coulter Co., Miami, FL) and percentages of each cell-cycle phase were determined with a Multicycle Cell-Cycle Analysis program (Coulter Co.).

Western blot analyses.
Protein levels of p21Waf1 and p27Kip1 in M6 and Pr14 cells treated with lovastatin for 24 or 48 h were determined by western blot. Cells were incubated with the lysis buffer on ice for 45 min. Extracts were then cleared by centrifugation, and protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA). Whole-cell lysates (20 µg) were fractionated through Tris–glycine gels under reducing conditions and transferred onto nitrocellulose or PVDF membranes (Immobilon-P; Millipore, Bedford, MA). After blocking with 3% bovine serum albumin, blots were incubated for 1 h at room temperature with anti-p21 (C-19) or anti-p27 (F-8) antibodies from Santa Cruz Biotechnology or actin (N350) from Amersham Life Science, Arlington Heights, IL. The blots were then washed and incubated for 1 h at room temperature with an appropriate horseradish peroxidase-conjugated secondary antibody. Proteins were visualized using enhanced chemiluminescence (NEN Life Science, Boston, MA).

Microarrays.
M6 cells (1x106) were plated into 100 mm dishes 1 day prior to treatment. Cells were treated with 10 µM of lovastatin (n = 4) or control vehicle ethanol (n = 4) for 24 h. RNA was isolated using guanidine isothiocyanate method. RNA (20 mg) from control and lovastatin-treated cells were labeled and hybridized as described previously (16) except that the reactions were purified using Microcon YM-30 columns. The Incyte GEM1 set of cDNA clones (8700 features) and the mouse oncochip of the National Cancer Institute array (2700 features) were produced by the National Cancer Institute array facility. Each sample was tested on the Gem1 and mouse oncochip arrays. The array slides were scanned using a Genepix scanner (Axon Instruments, Foster City, CA) using suitable PMT voltages in each channel at a resolution of 10 µ. The control vehicle-treated cells were the internal reference RNA and were labeled using Cyanine 3-dUTP(Cy3) and the lovastatin-treated cells were labeled with Cyanine 5-dUTP (Cy5). Image analysis and the calculation of average foreground signal adjusted for local channel-specific background was performed using GenePix software. All statistical analyses were performed using the S+ package. Spots with signal intensities in both channels <100 were excluded. Induced mRNA levels were determined using NCI mAdb array bioinfomatics software (nciarray.nci.nih.gov/).

In vivo studies
A total of 92 C3(1)/TAg transgenic mice (42 females and 39 males at 3 months of age and 30 females at 4 months of age) were used in this study. Mammary and prostate tumor progression in the transgenic mice follows a predictable time course: atypical hyperplasias (low-grade MIN) of the mammary ducts are noted at 2 months of age in the females, become nodular lesions resembling human ductal carcinoma in situ (high-grade MIN) (11) by ~3 months of age, and progress to adenocarcinomas by 4 months of age. Most of the female transgenic mice die with mammary cancer between 6 and 7 months of age. In the transgenic males, low-grade prostatic intraneoplasia (PIN) is observed in the prostate at 2–3 months of age, whereas high-grade PIN is found by 5 months of age. A progressive increase in the number of PIN lesions is observed with age (9). Prostate carcinomas, which appear to arise from PIN lesions, are found in the prostate after ~7 months of age (10). In order to assess the effect of lovastatin treatment on the progression of MIN and PIN lesions, male and female mice were treated with lovastatin beginning at 3 months of age. Female mice were randomly divided into three cohorts of 15–16 mice and male mice were randomly divided into two groups of 20 and 25 mice. The two treated female groups received either 25 mg/kg lovastatin intraperitoneally (i.p.) three times per week for 4 weeks or 50 mg/kg lovastatin i.p. three times per week for 12 weeks. Control mice were injected with vehicle alone. As male mice receiving a dose of 50 mg/kg lovastatin i.p. three times per week died for unknown reasons after 1 week in a separate study (data not shown), a dosage of 50 mg/kg once a week for 16 weeks was chosen for male mice in this study. Control mice were treated with vehicle alone. The doses used were based upon the results of a short-term study where doses ranged from 0 to 50 mg/kg administered i.p. three times per week (12,14,17). Animal weights were recorded weekly and used to determine appropriate doses each week. Transgenic female mice were killed following 4–12 weeks of treatment with lovastatin. Male transgenic mice were killed after 16 weeks of lovastatin treatment. Axillary and femoral mammary glands from the females and the entire prostate with seminal vesicles of the males were immediately removed and fixed in 4% paraformaldehyde, embedded in paraffin, cut at a thickness of 4-µm and stained with hematoxylin and eosin (H&E) for histopathological examination. As the mammary tumors in the females were fused and irregularly shaped by 24 weeks of age, tumors were measured with a caliper and the calculated tumor volume was determined using the formula: maximum diameterx(minimum diameter)2x0.4 (18).

Cell proliferation.
Female mice that received 4 weeks of lovastatin treatment were injected with 50 mg/kg of 5-bromo-2'-deoxyuridine (BrdU; Sigma, St Louis, MO) i.p. 1 h prior to death. Immunohistochemical staining for BrdU was performed on paraffin-embedded sections of the mammary glands to access DNA synthesis and cell proliferation. The numbers of cells incorporating BrdU into DNA were counted in a population of up to 3000 mammary ductal cells within the same type of histopathologic lesion using an ocular micrometer disk (Fisher Scientific, Pittsburgh, PA) and expressed as a percentage of BrdU-positive cells.

Analyses of apoptosis.
Levels of apoptosis in mammary lesions from females treated with lovastatin for 4 weeks, were quantitatively analyzed in 4-µm sections from paraffin-embedded tissues using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) method (Apop Tag; Oncor, Gaithersburg, MD). The number of apoptotic cells in paraffin sections was counted as described above for BrdU staining. Levels of apoptosis were expressed as a percentage of the total cells counted. Sections from testes were used as a positive control. As this technique may also stain cells undergoing necrotic death, areas of necrosis and regions immediately adjacent to necrotic areas within mammary carcinomas were excluded from the quantification.

Statistical analysis
To evaluate dose–response effects, all data (except for flow cytometry) were subjected to the analysis of variance and differences between means by the Scheffe’s t-test. Data from flow cytometric analyses were evaluated comparing untreated and 10 µM lovastatin-treated groups using a two-sided Student’s t-test. The incidences of histopathological findings were analyzed by the two-sided Fisher’s exact probability test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro studies
Effects of lovastatin on cell growth and apoptosis.
In order to determine the minimum concentration of lovastatin, which inhibits the growth of M6 and Pr14 cells, a dose–response study was conducted. As 10 µM lovastatin induced a 78% inhibition of cell growth for M6 cells (Figure 1AGo) and 82% inhibition for Pr14 cells (Figure 1BGo) after 48 h of treatment, a dose of 10 µM was chosen for further in vitro studies. A time-course of cell growth inhibition by 10 µM lovastatin was performed (Figure 1C and DGo). Lovastatin treatment of M6 cells resulted in a significant inhibition of cell growth after 48 and 72 h of treatment of M6 cells (Figure 1CGo). However, lovastatin treatment of Pr14 cells for 24–72 h appeared to actually reduce the number of cells (Figure 1DGo). The rate of growth inhibition by lovastatin compared with the controls was 38, 53 and 73% for M6 cells and 29, 88 and 98% for Pr14 cells after 24, 48 and 72 h of treatment, respectively (Figure 1C and DGo). As seen in Figure 2Go, treatment of M6 and Pr14 cells with lovastatin resulted in reduced cell numbers and numerous dead cells as compared with non-treated M6 cells (Figure 2BGo). In addition, the lovastatin-treated cells became rounder and less adherent to the culture plate (Figure 2Go). DNA fragmentation was observed after 24 h of exposure to lovastatin and increased with longer periods of treatment (Figure 2EGo, upper). As SV40 TAg has been shown to inactivate p53 through SV40 binding to p53 protein (19), transcriptional levels of p53 were analyzed. An elevation in the transcriptional levels of p53 was not associated with the induction of apoptosis (Figure 2EGo, lower), suggesting that lovastatin does not alter p53 gene expression in these cell lines.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1. Inhibition of proliferation by lovastatin in murine mammary (A) and prostate (B) carcinoma cells after 48 h. Time course of inhibition of cell growth in mammary (C) and prostate (D) carcinoma cells treated with 10 µM lovastatin. Lovastatin significantly inhibited both types of cancer cells. Data presented are mean ± SD values. Significantly different from control values at P <0.05 (*) and P < 0.01 (**).

 



View larger version (207K):
[in this window]
[in a new window]
 
Fig. 2. Lovastatin-induced reduction in cell numbers containing dead (orange) cells in mammary (B) and prostate (D) carcinoma cells as compared with control cells (A and C). Lovastatin-treated cells appeared rounder, and easily detached (B and D). Stained with acridine orange and ethidium bromide, and visualized by fluorescence microscopy (A–D). Lovastatin-induced apoptotic DNA fragmentation in mammary (M6) and prostate (Pr14) carcinoma cells after lovastatin exposure for 24 h on a modified agarose gel (E, upper). p53 mRNA levels were not elevated after 48 and 72 h of lovastatin exposure by northern blot (E, lower), although cells were undergoing apoptosis. GAPDH was used as an internal control.

 
Cell-cycle distribution.
Lovastatin induced a significant elevation in G1 arrest in both M6 and Pr14 cells as compared with untreated cells as measured by flow cytometry (Table IGo). Lovastatin also significantly reduced the S phase fractions of both cell lines and the G2/M fraction of the Pr-14 cells (Table IGo). Western blot analyses demonstrated that lovastatin increased p21 and p27 levels in both types of cancer cells after 24 or 48 h of exposure (Figure 3Go).


View this table:
[in this window]
[in a new window]
 
Table I. Effect of lovastatin on cell cycle distribution in mammary and prostrate carcinoma cells

 


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 3. Increased protein expression of p21Waf1 and p27Kip1 in mammary (M6) and prostate (Pr14) carcinoma cells treated with 10 µM lovastatin for up to 48 h as demonstrated by western blotting.

 
Microarrays.
Microarray analyses demonstrated few changes in mRNA transcriptional levels between M6 cells treated with lovastatin and control vehicle at 24 h (Figure 4Go). One gene had a 2-fold induction in several arrays. The sequence of this EST was similar to pre B-cell leukemia transcription factor 1 (Pbx-1).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. Microarray mean intensities of mammary (M6) carcinoma cells treated with 10 µM lovastatin compared with cells treated with control vehicle for 24 h for 8.7 K array features. Mean array intensity was 0.977 ± 0.207 (SD values). Little difference is observed between treated and untreated cells.

 
In vivo studies
Body weight curve.
Data on body weights of transgenic mice treated with lovastatin are shown in Figure 5AGo (females) and B (males). No statistical differences in body weights between control and lovastatin-treated groups were observed throughout the experiment in either sex. No compound-related clinical morbidity or mortality was observed.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5. Body weights of females (A) and males (B) in C3(1)/TAg transgenic mice treated with lovastatin: control (open circle), 25 (open square) and 50 mg/kg (closed triangle). No significant differences between control and lovastatin-treated groups were observed.

 
Lovastatin inhibits MIN formation.
Four mice from each group were killed after 4 weeks of treatment. Mammary lesions were classified into three types: atypical hyperplasia (also referred to as low grade MIN), high-grade MIN and adenocarcinoma. High-grade MIN lesions (referred to previously as nodular atypical hyperplasia) (11) were considered to be pre-neoplastic lesions (9). The multiplicity of mammary lesions in mice treated with lovastatin for 4 weeks is shown in Figure 6AGo. Mice treated with 50 mg/kg lovastatin showed a significant reduction in numbers of MIN lesions as compared with the values in control mice. Treatment with lovastatin at the lower does of 25 mg/kg did not result in a significant reduction in MIN lesions. There were no differences in the incidence of mammary carcinomas after 4 weeks of treatment between control and lovastatin-treated groups.



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 6. Effects of lovastatin on mammary carcinogenesis in C3(1)/TAg transgenic female mice—sacrificed at early (A–C) and late (D) stage mammary carcinogenesis. Treatment with 50 mg/kg lovastatin for 4 weeks significantly reduced the number of MIN but not carcinomas (A). Levels of apoptosis were significantly elevated in atypical hyperplasias and MIN lesions of mice receiving 50 mg/kg lovastatin (B). DNA synthesis in these lesions was similar among groups (C). Tumor volume tended to be lower in mice receiving lovastatin for 12 weeks, but this was not statistically significant (D). Data presented are mean ± SD values. Significantly different from control values at P < 0.05 (*) and P < 0.01 (**).

 
Apoptosis and cell proliferation in early mammary lesions.
Levels of apoptosis in mammary lesions of mice treated with lovastatin for 4 weeks are shown in Figure 6BGo. The levels of apoptosis were significantly increased in atypical hyperplasias and MIN lesions of mice receiving 50 mg/kg lovastatin as compared with the values of control mice. There was no significant difference in the levels of apoptosis between control mice and mice treated with 25 mg/kg lovastatin. BrdU labeling indices were not statistically different between the control and lovastatin-treated groups (Figure 6CGo).

Effect of long-term lovastatin on mammary tumorigenesis.
Eight out of 11 (73%) mice in the control group were alive after 4 months of treatment, compared with 11 out of 12 (92%) in the group given 25 mg/kg of lovastatin, and 11 out of 12 (92%) in the group given 50 mg/kg of lovastatin. Death was performed for tumors >2 cm in diameter. As transgenic mice at 6 months of age have multiple invasive carcinomas that coalesce, it was not possible to quantify MIN and invasive carcinoma lesions at this age. However, mammary tumor volumes were determined and tended to decrease in female mice receiving 25 and 50 mg/kg as compared with the values for control animals (Figure 6DGo). The lack of statistical significance between these groups may be due to the large variation within the relatively small groups of animals. Although lung metastasis was not observed in single cut lung sections in any group, this study cannot address whether lovastatin might inhibit the formation of metastases.

Effect of lovastatin on prostate lesion formation.
Lovastatin did not improve survival in male mice at 7 months of age after receiving treatment for 4 months. Nine out of 20 mice (45%) in the control group were alive at 7 months of age whereas 12 out of 25 (48%) remained alive in the group treated with 50 mg/kg of lovastatin. Animals died as a result of urinary obstruction due to tumors of the prostate and/or bulbourethral glands (20). Prostate lesions were divided into three types: low-grade PIN, high-grade PIN and carcinoma (10). The incidence and multiplicity of lesions in the ventral and dorso-lateral prostate after 4 months of lovastatin treatment are shown in Figure 7A and BGo, respectively. No significant differences in prostate lesion development were observed between the lovastatin-treated and control groups.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 7. Effects of lovastatin on prostate carcinogenesis in C3(1)/TAg transgenic male mice. Incidence (A) and multiplicity (B) of the prostate lesions. No significant differences were observed between control and lovastatin-treated mice. Vertical lines in (A) indicate confidence intervals; error bars in (B) indicate standard deviation.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lovastatin is a drug that has been widely used clinically to reduce hypercholesterolemia and is regarded as generally safe. Lovastatin inhibits HMG-CoA reductase, which is the rate-limiting enzyme of the cholesterol biosynthesis pathway (21). The inhibition of the cholesterol biosynthesis pathway by lovastatin not only blocks mevalonate synthesis (the product of HMG-CoA reductase), but also prevents the farnesylation and geranylgeranylation (intermediate products of the cholesterol pathway) of several signal transduction proteins, such as Ras and G proteins (22). Buchwald has hypothesized that an important feature of malignant transformation is loss of the cholesterol feedback inhibition mechanism that regulates cholersterol synthesis (23). These mechanisms of action may have potential impact in the inhibition of pathways involved in oncogenesis. Supporting this hypothesis are data from a 5-year clinical safety and efficacy study of lovastatin in which there appeared to be a reduction in cancer deaths in the group receiving lovastatin (24).

Several studies have demonstrated an antitumor activity of lovastatin in animal models. Maltese et al. (25) found that lovastatin suppressed neuroblastoma growth in mice, and that these tumors had few or no mitotic figures. Simvastatin, another inhibitor of HMG-CoA and synthetic derivative of lovastatin, has anticarcinogenic activity on mammary tumorigenesis of irradiated rats (26), colon tumorigenesis in mice pre-treated with a colon carcinogen (27) and glioma intracerebrally inoculated in rats (28).

Tumor growth is inhibited in the skin carcinogenesis model of the SENCAR mouse when Ras function is inactivated by lovastatin through the blockade of Ras localization to the membrane (17). We have recently demonstrated that mammary tumor progression in the C3(1)/TAg model is associated with Ki-ras amplification and an elevation in MAP kinase activity (29). The lack of one normal Ki-ras allele retards early mammary tumor progression in this model (30). We hypothesized that lovastatin might inhibit tumorigenesis in this model by suppressing the Ras pathway. Although we have not determined whether ras amplification occurs in the prostate tumors of the C3(1)/TAg mice, a significant number of PIN lesions and prostate carcinomas contain ras mutations (10), whereas ras mutations in mammary tumors are not frequent (29). It appears that despite the expression of the same transgene, the selective pressures for genetic alterations differ during the mammary and prostate oncogenesis in these models. Based upon the known activities of lovastatin, we wished to explore whether lovastatin might have chemopreventive effects in mouse models of prostate and mammary cancer in which tumor stage-specific effects of lovastatin could be assayed.

The results of this study demonstrate that lovastatin significantly inhibits cell growth and induces apoptosis in both murine mammary and prostate carcinoma cells in vitro. Since lovastatin has been shown to inhibit cell growth with G1 arrest and reduced transition to the S and G2/M phases of the cell cycle (4,5,31) as well as induce apoptosis (6,7), we determined whether these effects could be induced in mammary and prostate carcinoma cells derived from transgenic tumors. Flow cytometric analyses showed that the inhibitory effect on cell proliferation by lovastatin, in both the mammary and prostate cells, appears to be due to G1 arrest and reduced DNA synthesis. Lovastatin has been shown to elevate levels of two key cell-cycle inhibitors, p21 (Waf1/Cip1) and p27 (Kip1) (32), leading to suppression of cell-cycle progression through their association with Cdks (33,34). In addition, lovastatin has been shown to reduce levels of Cdks 2 and 4 (5,34) and increase binding of p21 and p27 to Cdk2, resulting in a reduction of Cdk2 activity and G1 arrest in both normal mammary and breast tumor cell lines (31). Our results demonstrate that lovastatin leads to an increase in p21 and p27 protein levels in both the mammary and prostate cancer cell lines, as demonstrated by western blot analyses.

As M6 and Pr14 cells used in this study were derived from the C3(1)/SV40 TAg transgenic tumors, both p53 and pRb proteins were functionally inactivated in these cells by their binding to SV40 TAg (20,35). Although lovastatin induced apoptosis in these cells, we observed no increase in p53 transcription. These results suggest that the lovastatin-induced apoptosis occurs through a p53-independent mechanism. As 50% of human cancers have p53 mutations (36), the fact that the lovastatin induces a p53-independent apoptotic response may be highly relevant to inhibiting many human cancers. It has been reported previously that lovastatin increases p53 expression in normal human mammary cells (37) suggesting that lovastatin might have important anti-proliferative effects in cells without p53 mutations.

In the microarray analyses, no transcriptional changes >2-fold were found in mammary cancer cells following treatment with lovastatin for 12 or 24 h. It is possible that transcriptional changes might be occurring for genes not represented on the arrays, that transcriptional changes occur at other time-points, or that the changes occur in genes below the sensitivity of the microarray assay system. Cell death associated with RNA degradation occurred by 48 h in response to lovastatin. We have used arrays from the same production lot and in hybridizations performed at the same time to demonstrate striking differences in gene expression profiles in other experimental systems (3839) and, therefore, do not believe these results to be due to technical problems. Given the lack of significant changes induced by lovastatin at the RNA level, the effects of lovastatin appear to occur primarily through post-transcriptional mechanisms that may be further addressed using proteomic approaches.

Although we were able to demonstrate significant growth inhibitory effects by lovastatin in vitro, lovastatin appeared to exert only a modest effect in vivo in the C3(1)/SV40 TAg mice. Lovastatin inhibited the formation of MIN lesions in the mammary gland, with a tendency for reduced tumor formation and seemed to have no effect on prostate lesion development. This apparent discrepancy between the in vitro and in vivo results may be due to several factors.

It is possible that the dose and timing of administration was not sufficient in the present study to adequately inhibit tumor growth. For instance, BrdU labeling indices were evaluated in mice one day after lovastatin treatment but the lovastatin-induced G1 arrest and the inhibition of the S phase in mammary epithelial cells may not be sustained for 24 h, as peak levels of lovastatin in the blood occur by 2 h post-oral ingestion (40). To achieve continuous treatment of lovastatin, an osmotic pump to provide continuous dosing would be useful. Further trials will address these pharmacologic issues. It is also possible that the tumor inhibitory effects of lovastatin observed in vitro might be mitigated by epithelial-stromal interactions in vivo. Whereas lovastatin has an inhibitory effect directly on the tumor epithelial cells in vitro, it is possible that paracrine effects of stromal cells in the vicinity of the epithelial cells within the tumors may produce factors that overcome the inhibitory effects of lovastatin. Additionally, the effects of lovastatin may be modulated according to the background strain of the mouse studied.

Although there appears to be an apparent paradox in that there was a reduction in MIN lesions, but no decrease in palpable tumor formation, we believe that this is probably the result of lovastatin being given after a stage of tumorigenesis where a subset of early lesions had already committed to becoming aggressive, invasive carcinomas. Previous work from our laboratory has demonstrated that TAg expression is observed in a small number of mammary ductal cells by 3 weeks of age. In this study, lovastatin treatment was initiated at 12 weeks of age, providing ~9 weeks of TAg expression without lovastatin. It is quite possible that some of the lesions, which developed from the early TAg-expressing cells were fully transformed and able to progress to invasive carcinomas despite the presence of lovastatin. Lovastatin was, however, able to retard lesion development in TAg-expressing cells, which had not reached this critical state of transformation, thus, accounting for the reduction in MIN lesions.

The present results suggest that as lovastatin exerts an inhibitory effect on early lesions of mammary oncogenesis, this compound may be useful for chemoprevention of mammary cancer and possibly as an adjuvant to the therapy of breast cancer with p53 mutations. However, as lovastatin did not inhibit prostate lesion development, the inhibitory effects of lovastatin may be tissue-specific.


    Notes
 
4 To whom correspondence should be addressed Email: jegreen{at}nih.gov Back


    Acknowledgments
 
We thank Lisa Birely, Darlene Reever and Dan Longston of the in vivo Carcinogenesis Program in NCI-FCRDC for animal technical assistance. We also thank Mr T.Ueno at the Central Research Center of Osaka Medical College for assistance with flow cytometric analysis.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Parker,S.L., Tong,T.A., Bolden,S. and Wingo,P.A. (1996) Cancer statistics. CA Cancer J. Clin., 46, 5–27 [Comment. (1996) CA Cancer J. Clin., 46, 3–4].[Abstract/Free Full Text]
  2. Harris,J.R., Lippman,M.E., Veronesi,U. and Willett,W. (1992) Breast cancer. N. Engl. J. Med., 327, 319–328, 390–398.[ISI][Medline]
  3. Goldstein,J.L. and Brown,M.S. (1990) Regulation of the mevalonate pathway. Nature, 343, 425–430.[CrossRef][ISI][Medline]
  4. Keyomarsi,K., Sandoval,L., Band,V. and Pardee,B. (1991) Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. Canncer Res., 51, 3602–3609.
  5. Gray-Bablin,J., Rao,S. and Keyomarsi,K. (1997) Lovastatin induction of cyclin-dependent kinase inhibitors in human breast cells occurs in a cell cycle-independent fashion. Cancer Res., 57, 604–609.[Abstract]
  6. Borner,M.M., Myers,C.E., Sartor,O., Sei,Y., Toko,T., Trepel,J.B. and Schneider,E. (1995) Drug-induced apoptosis is not necessarily dependent on macromolecular synthesis or proliferation in the p53-negative human prostate cancer cell line PC-3. Cancer Res., 55, 2122–2128.[Abstract]
  7. Padayatty,S.J., Marcelli,M., Shao,T.C. and Cunningham,G.R. (1997) Lovastatin-induced apoptosis in prostate stromal cells. J. Clin. Endocrinol. Metab., 82, 1434–1439.[Abstract/Free Full Text]
  8. Maroulakou,I.G., Anver,M., Garrett,L. and Green,J.E. (1994) Prostate and mammary adenocarcinoma in transgenic mice carrying a rat C3 (1) simian virus 40 large tumor antigen fusion gene. Proc. Natl Acad. Sci. USA, 91, 11236–11240.[Abstract/Free Full Text]
  9. Shibata,M.-A., Maroulakou,I.G., Jorcyk,C.L., Gold,L.G., Ward,J.M. and Green,J.E. (1996) p53-independent apoptosis during mammary tumor progression in C3 (1)/SV40 large T antigen transgenic mice: suppression of apoptosis during the transition from preneoplasia to carcinoma. Cancer Res., 56, 2998–3003.[Abstract]
  10. Shibata,M.-A., Ward,J.M., Devor,D.E., Liu,M.-L. and Green,J.E. (1996) Progression of prostatic intraepithelial neoplasia to invasive carcinoma in C3 (1)/SV40 large T antigen transgenic mice: histopathological and molecular biological alterations. Cancer Res., 56, 4894–4903.[Abstract]
  11. Green,J.E., Shibata,M.-A., Yoshidome,K. et al. (2000) The C3 (1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma. Oncogene, 19, 1020–1027.[CrossRef][ISI][Medline]
  12. Alonso,D.F., Farina,H.G., Skilton,G., Gabri,M.R., de Lorenzo,M.S. and Gomez,D.E. (1998) Reduction of mouse mammary tumor formation and metastasis by lovastatin, an inhibitor of the mevalonate pathway of cholesterol synthesis. Breast Cancer Res. Treat., 50, 83–93.[CrossRef][ISI][Medline]
  13. Hawk,M.A., Cesen,K.T., Siglin,J.C., Stoner,G.D. and Ruch,R.J. (1996) Inhibition of lung tumor cell growth in vitro and mouse lung tumor formation by lovastatin. Cancer Lett., 109, 217–222.[CrossRef][ISI][Medline]
  14. Jani,J.P., Specht,S., Stemmler,N., Singh,S.V., Gupta,V. and Katoh,A. (1993) Metastasis of B16F10 mouse melanoma inhibited by lovastatin, an inhibitor of cholesterol biosynthesis. Invasion Metast., 13, 314–324.[Medline]
  15. Jorcyk,C.L., Liu,M.-L., Shibata,M.-A., Maroulakou,I.G., Komschlies,K.L., McPhaul,M.J., Resau,J.H. and Green,J.E. (1998) Development and characterization of a mouse prostate adenocarcinoma cell line: ductal formation determined by extracellular matrix. Prostate, 34, 10–22.[CrossRef][ISI][Medline]
  16. Hegde,P., Qi,R., Abernathy,R., Gay,C., Dharap,S., Gaspard,R., Earle-Hughes,J., Snesrud,E., Lee,N.H. and Quackenbush,J. (2000) A concise guide to cDNA microarray analysis. Biotechniques, 29, 548–562.[ISI][Medline]
  17. Khan,S.G., Saxena,R., Bickers,D.R., Mukhtar,H. and Agarwal,R. (1995) Inhibition of ras p21 membrane localization and modulation of protein kinase C isozyme expression during regression of chemical carcinogen-induced murine skin tumors by lovastatin. Mol. Carcinogen., 12, 205–212.[ISI][Medline]
  18. Fueyo,J., Gomez-Manzao,C., Yung,W.K.A., Liu,T.J., Alemany,R., McDonnell,T.J., Shi,X., Rao,J.S., Levin,V.A. and Kyritsis,A.P. (1998) Overexpression of E2F1 in glioma triggers apoptosis and suppress tumor growth in vitro and in vivo.Nature Med., 4, 685–690.[ISI][Medline]
  19. Dyson,N., Buchkovich,K., Whyte,P. and Harlow,E. (1989) The cellular 107K protein that binds to adenovirus E1A also associates with the large T antigens of SV40 and JC virus. Cell, 58, 249–255.[ISI][Medline]
  20. Shibata,M.-A., Jorcyk,C.J., Devor,D.E., Yoshidome,K., Rulong,S., Resau,J., Roche,N., Roberts,A.B., Ward,J.M. and Green,J.E. (1998) Altered expression of transforming growth factor bs during urethral and bulbourethral gland tumor progression in transgenic mice carrying the androgen-responsive C3 (1) 5' flanking region fused to SV40 large T antigen. Carcinogenesis, 19, 195–205.[Abstract]
  21. Alberts,A.W., Chen,J., Kuron,G. et al. (1980) Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent. Proc. Natl Acad. Sci. USA, 77, 3957–3961.[Abstract]
  22. Maltese,W.A. (1990) Posttranslational modification of proteins by isoprenoids in mammalian cells. FASEB J., 4, 3319–3328.[Abstract/Free Full Text]
  23. Buchwald,H. (1992) Cholesterol inhibition, cancer and chemotherapy. Lancet, 339, 1154–1156.[CrossRef][ISI][Medline]
  24. Groups,T.L.S. (1993) Lovastatin 5-year safety and efficacy study. Arch. Intern. Med., 153, 1079–1087.[Abstract]
  25. Maltese,W.A., Defendini,R., Green,R.A., Sheridan,K.M. and Donley,D.K. (1985) Suppression of murine neuroblastoma growth in vivo by mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J. Clin. Invest., 76, 1748–1754.[ISI][Medline]
  26. Inano,H., Suzuki,K., Onoda,M. and Wakabayashi,K. (1997) Anti-carcinogenic activity of simvastatin during the promotion phase of radiation-induced mammary tumorigenesis of rats. Carcinogenesis, 18, 1723–1727.[Abstract]
  27. Narisawa,T., Fukaura,Y., Terada,K., Umezawa,A., Tanida,N., Yazawa,K. and Ishikawa,C. (1994) Prevention of 1,2-dimethylhydrazine-induced colon tumorigenesis by HMG-CoA reductase inhibitors, pravastatin and simvastatin, in ICR mice. Carcinogenesis, 15, 2045–2048.[Abstract]
  28. Soma,M.R., Baetta,R., de Renzis,M.R., Mazzini,G., Davegna,C., Magrassi,L., Butti,G., Pezzotta,S., Paoletti,R. and Fumagalli,R. (1995) In vivo enhanced antitumor activity of carmustine [N,N'-bis (2-chloroethyl)-N-nitrosourea] by simvastatin. Cancer Res., 55, 597–602.[Abstract]
  29. Liu,M.-L., Von Lintig,F.C., Liyanage,M., Shibata,M.-A., Jorcyk,C.L., Ried,T., Boss,G.R. and Green,J.E. (1998) Amplification of Ki-ras and elevation of MAP kinase activity during mammary tumor progression in C3 (1)/SV40 Tag transgenic mice. Oncogene, 17, 2403–2411.[CrossRef][ISI][Medline]
  30. Liu,M.-L., Shibata,M.-A., Von Lintig,F.C., Wang,W., Boss,G.R. and Green,J.E. (2001) Haploid loss of Ki-ras delays mammary tumor progression in C3 (1)/SV40 Tag transgenic mice. Oncogene, 20, 2044–2049.[CrossRef][ISI][Medline]
  31. Rao,S., Lowe,M., Herliczek,T.W. and Keyomarsi,K. (1998) Lovastatin mediated G1 arrest in normal and tumor breast cells is through inhibition of CDK2 activity and redistribution of p21 nad p27, independent of p53. Oncogene, 17, 2393–2402.[CrossRef][ISI][Medline]
  32. Harper,J.W. and Elledge,S.J. (1996) Cdk inhibitors in development and cancer. Curr. Opin. Gene Dev., 6, 56–64.[ISI][Medline]
  33. Dulic,V., Stein,G.H., Far,D.F. and Reed,S.I. (1998) Nuclear accumulation of p21 Cip1 at the onset of mitosis: a role at the G2/M-phase transition. Mol. Cell. Biol., 18, 546–557.[Abstract/Free Full Text]
  34. Hengst,L. and Reed,S.I. (1996) Translational control of p27Kip1 accumulation during the cell cycle. Science, 271, 1861–1864.[Abstract]
  35. Ludlow,J.W. (1993) Interactions between SV40 large-tumor antigen and the growth suppressor proteins pRB and p53. FASEB J., 7, 866–871.[Abstract/Free Full Text]
  36. Greenblatt,M.S., Bennett,W.P., Hollstein,M. and Harris,C.C. (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res., 54, 4855–4878.[ISI][Medline]
  37. Gudas,J.M., Oka,M., Diella,F., Trepel,J. and Cowan,K.H. (1994) Expression of wild-type p53 during the cell cycle in normal human mammary epithelial cells. Cell Growth Differ., 5, 295–304.[Abstract]
  38. Desai,K., Xiao,N., Wang,W. et al. (2002) Defining cancer gene expression patterns of mouse models of human breast cancer: initiating oncogenic event determines tumor signature. Proc. Natl Acad. Sci. USA, 99, 6967–6972.[Abstract/Free Full Text]
  39. Calvo,A., Nianqing,X., Powell,J., Kang,J., Best,C., Emmert-Buck,M., Jorcyk,C. and Green,J.E. (2002) Alterations in Gene Expression Profiles During Prostate Cancer Progression: functional correlations to tumorigenicity and down-regulation of selenoprotein-P in mouse and human tumors. Cancer Res., 62, 5325–5335.[Abstract/Free Full Text]
  40. Duggan,D.E., Chen,I.W., Bayne,W.F., Halpin,R.A., Duncan,C.A., Schwartz,M.S., Stubbs,R.J. and Vickers,S. (1989) The physiological disposition of lovastatin. Drug Met. Dispos., 17, 166–173.[Abstract]
Received June 28, 2002; revised November 13, 2002; accepted November 17, 2002.