Affiliations of authors: Women's Cancers Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD (DP, DOH, TO, CEH, MS, PSS); Laboratory Animal Sciences Program, SAIC, Frederick, MD (JJ); Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD (WDF); Developmental Therapeutics Program, National Cancer Institute, Bethesda, MD (MH); Laboratory of Biosystems and Cancer, Center for Cancer Research, National Cancer Institute, Bethesda, MD (SH, DB); Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, Bethesda, MD (SMS); Surgical Pathology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD (MJM)
Correspondence to: Patricia S.Steeg, PhD, National Institutes of Health, Bldg. 10, Rm. 2A33, Bethesda MD 20892 (e-mail: steegp{at}mail.nih.gov).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nm23 was the first metastasis suppressor gene identified (4,5). Subsequently, eight members of the Nm23 gene family (Nm23-H1 through -H8) have been reported. Although the eight genes share homology, the in vivo data show that Nm23-H1 is most likely to function as a metastasis suppressor gene (6). Eleven independent studies have confirmed that restoration of Nm23 expression reduces metastasis of breast, colon, prostate, oral squamous cell carcinoma, and melanoma tumor cell lines to the lymph nodes, lungs, and liver (5,716). In vitro correlates of elevated Nm23-H1 expression in breast carcinoma cell lines include reduced soft-agar colony formation, reduced cell motility, reduced invasion, and the induction of morphological and biochemical aspects of differentiation in three-dimensional culture (7,1720).
For both tumor suppressor genes and metastasis suppressor genes, when the gene for the wild-type protein is lost, a major challenge lies in how to restore its suppressive function to a metastatic cancer cell. If the suppressive function of the protein can be identified and mapped to a single, specific domain of the protein, then screening methods may be able to identify a compound whose activity can replace that of the suppressor gene (21). However, many suppressor proteins have multiple biochemical functions and functional domains, all of which may contribute to the overall phenotype. Nm23-H1 is a case in point: This protein interacts with several different proteins, exerts DNA regulatory functions, and exhibits a histidine kinase activity, all of which may contribute to its suppression of metastasis. For suppressor proteins of this type, multiple specific replacement compounds would need to be identified.
Although allelic deletion of Nm23-H1 does occur in human tumors, most poor-prognosis tumors are associated with reduced Nm23-H1 expression rather than with its allelic deletion or mutation, making reexpression of Nm23 in such tumors an attractive therapeutic option (22). We and others have identified compounds that stimulate metastatic tumor cells to reexpress Nm23-H1 in vitro (2327). In addition, we have found that the Nm23-H1 promoter contains a cassette of mammary-specific transcription factor binding sites regulated by glucocorticoid response elements that contribute to Nm23-H1 expression (28). Indeed, dexamethasone, prednisolone, and other glucocorticoids elevated Nm23-H1 expression in metastatic MDA-MB-435 and -231 breast carcinoma cell lines in vitro in the presence of charcoal-stripped medium (29), which contains no endogenous corticosteroids. High-dose inhibition of Nm23-H1 was observed, suggesting that these compounds are relevant to physiologic but not pharmacologic elevation of Nm23-H1 expression (29).
Another glucocorticoid, medroxyprogesterone acetate (MPA), has also been shown to elevate Nm23-H1 expression (29). MPA has a long clinical history. It is a progestin that is found at low doses in the contraceptive Depo-Provera (30) and is combined with estrogen in hormone replacement therapy. MPA has been tested at high doses as a single agent or in combination regimens as a hormonal treatment for advanced breast and uterine cancers [reviewed in (31)]. Although some patient responses were observed in clinical trials, an optimum dose and schedule were not determined. Two of the MPA clinical trials (32,40) have suggested that a longer course of MPA treatment would increase its clinical benefit in postmenopausal women. These two trials randomly assigned a total of 950 patients to chemotherapy (cytoxan, methotrexate, and 5-fluoruracil [CMF] in one trial and cytoxan, adriamycin, and 5-fluoruracil [CAF] in the other), with or without MPA. MPA was given for a 6-month time course after either CMF or CAF, compared with other trials, which limited MPA treatment to several weeks (31). Administration of MPA was divided into two phases, an induction phase of 500 mg daily for 28 days followed by a maintenance phase of 500 mg twice weekly for 6 months. After 12 and 13 years of follow-up, respectively, the MPA-treated postmenopausal subsets of patients had improved disease- and metastasis-free survival compared with patients who did not receive MPA (CAF, P = .01; CMF, P = .06 for node-negative and P = .002 for node-positive patients) and, for patients in the CAF ± MPA trial, longer overall survival (P = .02) (32,40).
Paradoxically, patient responses were not well correlated with progesterone receptor (PR) expression (3234). This lack of association made better sense years after the trials commenced with reports that MPA can interact not only with PR but also with the androgen receptor (35) and, as a glucocorticoid, with the glucocorticoid receptor (GR)the latter in both traditional (36) and nontraditional, i.e., DNA bindingindependent (3739), pathways. MPA elevates Nm23-H1 expression of the MDA-MB-435 and MDA-MB231 metastatic breast carcinoma cell lines two- to fourfold in vitro in culture medium supplemented with 10% fetal bovine serum (29). Both cell lines are ER and PR negative, and the effect of MPA on Nm23-H1 expression involves a nontraditional, GR-dependent molecular pathway that is regulated posttranslationally (29).
In this article, we characterized the effect of MPA on metastatic colonization of an ER-negative, PR-negative, GR-positive MDA-MB-231 breast carcinoma subline both in vitro (using soft-agar colony-forming assays) and in vivo (in a mouse model). One goal of the in vivo studies was to use MPA doses that are achievable in humans. To this end, we conducted a nine-arm pharmacokinetic study to determine an MPA dose and treatment schedule that would yield serum MPA levels in mice comparable to those attained in women in the clinical trials. For the in vivo metastasis experiments we adapted an in vivo experimental metastasis assay to focus on metastatic colonizationthe outgrowth of tumor cells from micrometastases to life-threatening lesions. Metastatic colonization is the last step in the metastatic process and has been hypothesized to represent the optimal therapeutic window of opportunity (1). For breast cancer patients with aggressive, lymph nodepositive disease, tumor cells may have already invaded past the primary tumor at time of diagnosis and surgery. Therefore, the outgrowth of tumor cells to a detectable size in a distant organi.e., metastatic colonizationand angiogenesis represent the only parts of the metastatic process that are known to be incomplete in lymph nodepositive patients. We compared Nm23-H1 protein expression in pulmonary metastases and the incidence, number, and size of pulmonary metastases between MPA-treated and untreated mice.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MPA was purchased from Sigma (St. Louis, MO) and resuspended in 20% chloroform80% ethanol at 100 mM and subsequently diluted in 100% ethanol to 1 mM for use in in vitro experiments. Clinical grade MPA (Depo-Provera; Pharmacia & Upjohn, Kalamazoo, MI) was diluted to 20 mg/mL in 8.6 mM polyethylene glycol (PEG 3350; Sigma) and 0.15 M NaCl (vehicle) for use in in vivo experiments.
Cell Culture
A subline of human MDA-MB-231 cells, which were designated MDA-MB-231T cells (gift of Dr. Zach Howard, Laboratory of Immunoregulation, NCI), was chosen for its reliable in vivo experimental metastatic potential. MDA-MB-231T cells and MDA-MB-231 cells (American Type Culture Collection, Manassas, VA) were genotyped by the Core Genotyping Facility, NCI. The Applied Biosystems Profiler Plus kit was used to polymerase chain reaction (PCR) amplify tetranucleotide short tandem repeat loci and the amelogenin locus. The amelogenin locus was used to identify sex. Sixteen loci were genotyped to confirm the relationship between the different strains of the MDA-MB-231cell line. Four of these loci differed between MDA-MB-231T and MDA-MB-231 cells (Table 1, Supplementary Data). Vials of MDA-MB-231T cells that were confirmed negative for murine antigen protein were stored in the NCI animal facility repository. Cells were cultured in Dulbecco's minimal essential medium containing 10% fetal bovine serum.
|
The cDNA for human Nm23-H1 was inserted in reverse orientation into pcDNA3.1 (Invitrogen, Carlsbad, CA) at the BamH1 restriction site in the multiple cloning region. MDA-MB-231T cell clones stably expressing antisense Nm23-H1 were generated by transfection with Effectene (Qiagen, Valencia, CA) and selection in 1 mg/mL Geneticin (Invitrogen). Control cells were transfected with empty vector. Two individual stable clones of control and antisense Nm23-H1, respectively, were used for the experiments described herein.
Western Blot Analysis
Cells (2.5 x 105) were plated in 100-mm2 tissue culture dishes and incubated overnight. Cells were then incubated in MPA or ethanol (vehicle) for 72 hours. Cell lysates were obtained from cells treated with MPA or vehicle using cold RIPA buffer (20 mM TrisHCl [pH 8], 100 mM NaCl, 10% glycerol, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing complete mini EDTA-free protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) and phosphatase inhibitor cocktails 1 and 2 (Sigma). Cellular lysates (30 mg total protein) were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Western blot analysis was performed for antiNm23-H1 (1 : 500; Cymbus Chemicon, Temecula, CA), anti-tubulin (1 : 5000; Ab-1, Oncogene, Cambridge, MA), anti-ER (1 : 300; clone EMR02, NovoCastra, Newcastle upon Tyne, UK), anti-PR (1 : 500; Upstate, Chicago, IL) and anti-GR (1 : 100; Oncogene) overnight at 4 °C. Appropriate horseradish peroxidaseconjugated secondary antibodies were used and detected by autoradiography with LumiGlo Reagents (Cell Signaling, Beverly, MA).
Anchorage-independent Proliferation Assays
Anchorage-independent colony-forming assays in soft agar were performed according to the previously published protocol (29). In brief, MDA-MB-231T cells were plated in a layer of 0.3% noble agar containing the indicated concentrations of MPA. This layer was overlaid on a layer of 0.7% noble agar in 1.88-cm2 wells of a 24-well tissue culture plate. Cultures were grown at 37 °C in 5% CO2 for 3 weeks. Total numbers of colonies (>50 cells) per well were counted using a light microscope; triplicate wells were done for each condition.
Dose and Schedule Study
Mice (n = 180) were randomized to groups of 20 that received MPA in nine different combinations of dose, schedule, and route of administration (i.e., intramuscular or subcutaneous). In all cases, mice received 4 weeks of induction MPA and 8 weeks of maintenance MPA. Induction dosing consisted of either biweekly or daily MPA injections for 4 weeks, and maintenance dosing consisted of one injection every fourth week thereafter for 8 weeks. Terminal bleeds were collected from five mice of each group on days 7, 21, 49, and 56 after receipt of the first MPA dose. Blood from each group of five mice was pooled and centrifuged to separate the serum. Pooled serum samples were stored at 70 °C. Mammary fat pads were removed at necropsy from mice killed on days 7 and 56, preserved in Bouin's solution, fixed in formalin, and embedded in paraffin for immunohistochemistry. All in vivo experiments were performed in accordance with an approved animal use contract.
Determination of Serum MPA Levels
Fifty microliters of 0.1 mg/mL nortestosterone (internal standard) was added to 300 µL of each serum sample. Hexane (3 mL) was then added, and the samples were vortexed and centrifuged for 5 minutes. The organic layer was collected and evaporated under liquid nitrogen. The residue was reconstituted in 200 µL of mobile phase, and 100 µL was injected into a high-pressure liquid chromatography (HPLC) system. A C18 column (Phenomenex, Torrance, California) was used with a 75 : 25 : 0.03 methanol0.1 M sodium phosphate (pH 3.5)triethylamine mobile phase at a flow rate of 1.2 mL/minute with a run time of 12 minutes. The retention time of MPA was approximately 9.1 seconds. The intraday variation of the standard curve was 5.4%, and the interday variation was 2.9%.
Metastasis Assay
Two independent in vivo experiments were conducted. In Experiment 1, 6-week-old Nu/Nu mice (n = 100) were acclimated for 2 weeks and were then injected intravenously in the lateral tail vein with 5 x 105 MDA-MB-231T cells. Four weeks later, 90 mice were randomized to one of three treatment groups (30 mice per group). Each group received subcutaneous injections of vehicle (see Materials and Methods) or 2 mg or 4 mg MPA twice weekly for 4 weeks (induction phase), followed by a maintenance phase of the same dose 4 weeks later. In Experiment 2, 6-week-old Nu/Nu mice (n = 160) were acclimated for 2 weeks and were then injected intravenously with 5 x 105 MDA-MB-231T cells. Four weeks later, 150 mice were randomized to one of five treatment groups (30 mice per group). Four groups received subcutaneous injections of vehicle or MPA (0.5-mg, 1-mg, or 2-mg doses) twice weekly for 4 weeks (induction phase), followed by a maintenance phase of half the original dose every other week thereafter for 6 weeks. The fifth group was injected with 2 mg of MPA twice weekly for the entire 10-week treatment period.
Throughout both experiments, mice were weighed weekly and monitored for signs of ill health and labored breathing. Mice were killed if pathologic conditions unrelated to the study (e.g., breathing difficulties) developed. Mice from each group were killed at various time points for collection of serum (four mice per group) and flash-frozen tissue (six mice per group). Additional mice not assigned to a treatment group were killed at various time points to determine whether gross pulmonary metastases had developed (10 mice). All mice alive at the end of each experiment were killed and necropsied to quantitate gross metastases. Lungs, skin, mammary fat pads, and liver were preserved in Bouin's solution. An investigator who was blinded to the experimental arm counted gross pulmonary metastases and measured them with a ruler while viewing through a magnifying glass.
Immunohistochemistry
Lung tissue obtained at necropsy from five mice per treatment group in Experiment 2 that had multiple metastases was fixed in formalin, embedded in paraffin, and sectioned (6-µm sections). One randomly chosen section from each of the five mice per treatment group was stained for Nm23 protein using an affinity-purified polyclonal rabbit antiNm23-H1/H2 antibody (Cymbus). An isotype-matched control antibody (goat antirabbit immunoglobulin G; Santa Cruz Biotechnology, Santa Cruz, CA) was used on a second, independent section to control for nonspecific binding. In brief, sections were deparaffinized in xylene, dehydrated in 100% ethanol, rehydrated in 95% ethanol and phosphate-buffered saline (PBS), and blocked sequentially with hydrogen peroxide and goat serum. Antigen retrieval was accomplished by incubating the slides in 10 mM citrate buffer, pH 3.0, for 30 minutes at 37 °C. Slides were incubated with primary antibody (diluted 1 : 5 in PBS with 1% goat serum) in a humidified chamber overnight at room temperature. Staining was visualized using the Vectastain ABC kit and the DAB Substrate kit (Vector Laboratories, Burlingame, CA). Stained sections were examined under a microscope, and every visible metastasis in the tissue section was counted. Staining intensities were categorized as homogeneous low (i.e., staining of pulmonary metastases was equivalent in intensity to staining of the surrounding lung parenchyma); heterogeneous high (i.e., the pulmonary metastases contained some areas of staining higher in intensity than that in the parenchyma); and homogeneous high (i.e., staining in all areas of the pulmonary metastases was higher in intensity than that in the parenchyma).
Body Content Analysis
Fat weight, lean weight, bone mineral density (BMD), and bone mineral content (BMC) were determined for 10 mice per treatment arm from Experiment 2 using dual-energy X-ray absorptiometry (DXA) (GE Lunar Piximus II, Madison, Wisconsin). Necropsied carcasses were weighed and then placed on the specimen tray for scanning. Lean weight was calculated by subtracting fat weight from carcass weight. [Validation studies for estimates of body composition and bone characteristics using the GE Lunar Piximus Dual Energy X-ray Absorptiometer on mouse carcasses suggest that DXA is as effective for mice as it is for humans (41).]
Histopathologic Analysis of the Mammary Fat Pad
The fourth mammary fat pad of each animal in Experiment 2 was preserved at necropsy. A hematoxylin and eosinstained, formalin-fixed, paraffin-embedded section was examined by a pathologist who was blinded to experimental arm. Fat pads were examined for the presence of any pathologic conditions, i.e., hyperplasia, dysplasia, or carcinoma.
Statistical Methods
We used the Wilcoxon rank sum test to compare the number of total metastases and the number of metastases 3 mm or larger in any dimension in control mice with those values in mice treated with each dose of MPA. The 3-mm cutoff for large metastases was chosen arbitrarily after necropsy as a second measure of metastatic colonization. In addition, we used the JonckheereTerpstra trend test to evaluate whether the total number of metastases or the number of metastases 3 mm or larger increased with increasing MPA dose. We used the Wilcoxon rank sum test to compare the presence of any metastases, the total number of metastases, and the number of large metastases between control mice and mice treated with any dose of MPA. The fraction of all mice with any metastases was also evaluated across all dose levels within each experiment using the exact CochranArmitage test for trend as implemented in SAS Version 8 (SAS Institute, Cary, NC). The fraction of mice with large metastases, both among all mice and among mice with any metastases, was also evaluated using the exact CochranArmitage trend test.
Because both experiments contained a vehicle-treated control group as well as a group that received a 2-mg dose of MPA, the results for the 2-mg treatment groups from both experiments were compared within each of those two dose groups to determine if they were similar (P>.05), which would allow the data to be pooled to assess differences in metastatic colonization by dose. In the case of the number of metastases and number of large metastases, the Wilcoxon rank sum test was used. The fraction of any metastases that were 3 mm or larger was compared between experiments using the likelihood ratio test derived from logistic regression modeling, with adjustment for multiple comparisons by the Hochberg method as needed (42). Finally, the pooled results (number of metastases and number of large metastases) from the 2-mg dose from both experiments were compared with the pooled results from the control mice from both experiments by the methods described above. All P values are two-tailed and, except as stated above, are presented with no formal adjustment for multiple comparison. In view of the number of comparisons performed, P values between .01 and .05 were interpreted as being of borderline statistical significance, and P<.01 was considered statistically significant.
For the immunohistochemical analysis of Nm23 expression, the distribution of the number of metastases exhibiting homogeneous low, heterogeneous high, and homogeneous high Nm23 expression was analyzed in two ways. First, we compared the absolute number of metastases at each expression level between all mice in the control and each of the MPA dose groups using a Wilcoxon rank sum test. In addition, we created a severity index for each mouse within a group that was equal to: 1 x (number of metastases with homogeneous low expression) + 2 x (number of metastases with heterogeneous high expression) + 3 x (number of metastases with homogeneous high expression). The distribution of the severity indices was compared within each group, and a Wilcoxon rank sum test was used to compare severity indices between groups of mice. The P values are two-tailed and presented without adjustment for multiple comparisons. However, given the number of comparisons performed, P values between .01 and .05 are interpreted as being of borderline statistical significance, whereas P<.01 was considered statistically significant.
Finally, analysis of variance (ANOVA) and analysis of covariance were used to assess the effects of treatment on body composition and bone characteristics. ANOVA was used to determine body weightindependent effects of treatments. Analysis of covariance allowed the inclusion of weight as a covariate in the analysis (43).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MPA was previously reported to elevate Nm23-H1 expression in MDA-MB-231 breast carcinoma cells by two- to fourfold in vitro and to inhibit soft-agar colony formation by approximately 50% (29). We obtained similar results using the MDA-MB-231T subline (Fig. 1), which was chosen for its reliable in vivo experimental metastatic potential. The MDA-MB-231T cells were confirmed to be ER negative, PR negative, and GR positive by western blot analysis, and treatment with MPA did not alter this phenotype (data not shown). Nm23-H1 expression in MDA-MB-231T cells increased by approximately threefold after 72 hours of culture in 100 nM MPA (Fig. 1, A). Additionally, soft agar colony formation was reduced by 40%50% for MDA-MB-231T cells treated with MPA compared with untreated controls (Fig. 1, B). To examine the extent to which the increased Nm23-H1 expression accounts for the ability of MPA to suppress colonization, MDA-MB-231T cells were stably transfected with an antisense Nm23-H1 construct or an empty-vector control before treatment with MPA. Nm23-H1 expression was decreased three- to fourfold in the antisense transfectants as compared with control transfectants (Fig. 1, C). The antisense transfectants also did not undergo an increase in Nm23-H1 expression when treated with MPA (Fig. 1, D). Moreover, soft agar colony formation of antisense-expressing clones was not inhibited by MPA (Fig. 1, E). Thus, MPA appeared to affect anchorage-independent colony formation via its effect on Nm23-H1 expression.
|
A goal of the metastasis studies reported here was to use MPA doses that result in serum MPA levels in mice similar to those achievable in humans. Pharmacokinetic data exist from the clinical trials using MPA as hormonal therapy for breast cancer. In one trial, plasma concentrations of MPA were determined by HPLC in 79 patients with advanced or recurrent breast cancer who were treated orally with 600800 mg of MPA per day (44). Mean serum MPA concentrations were 60 ng/mL for complete responders and partial responders, 34 ng/mL for patients with stable disease, and 23 ng/mL for patients with progressive disease (P<.001). Similarly, among 129 patients treated orally with 4002000 mg of MPA per day, mean serum concentrations ranged from 46 ng/mL in patients with progressive disease to 4870 ng/mL in patients with complete or partial responses or stable disease (45). We carried out an extensive dose and schedule study to achieve a similar range of MPA plasma concentrations in mice. Although most clinical trials of MPA for breast cancer used oral or intramuscular dosing, we did not consider the oral route because of health concerns related to repeated oral gavage. We also did not supply the MPA in the mice's water because of concerns that analysis of water consumed would be open to artifacts from dripping bottles. The doseschedule study therefore compared intramuscular and subcutaneous injection routes. The induction phasemaintenance phase regimen reported in two clinical trials that showed improved disease-free survival in postmenopausal women with breast cancer was adapted for use in these experiments (32,40).
MPA plasma concentrations similar to those achieved in the human clinical trials were obtained by both intramuscular and subcutaneous dosing throughout the study period (Table 1). Serum MPA concentrations in mice that received subcutaneous injections 5 days per week were generally higher than those achieved in human clinical trials, whereas the serum MPA concentrations obtained with the twice-weekly dosing were more reflective of those achieved in the clinical trials. Therefore, for Experiment 1 we used subcutaneous injections of 2 and 4 mg MPA twice weekly throughout the 4-week induction phase and every 4 weeks throughout the 8-week maintenance phase. With these two doses, differences in plasma MPA concentration were seen mainly during the maintenance period (i.e., on days 49 and 56 after the initial MPA injection) (Table 1).
To determine whether these serum levels of MPA were biologically active, we investigated Nm23-H1 expression in normal mammary epithelial cells of mice treated twice weekly with the 2-mg and 4-mg doses of subcutaneous MPA. Immunohistochemical analysis of mammary fat pads collected at necropsy on day 7 showed that Nm23-H1 was localized to the luminal side of the mammary epithelial cells in untreated mice. In both treatment groups, Nm23-H1 expression was increased in both the mammary epithelial cells and the stroma relative to expression in untreated mice (Fig. 2 and data not shown). An isotype-matched control antibody showed no staining (data not shown). These data suggest that MPA stimulates expression of Nm23-H1 in normal mouse mammary epithelial cells in vivo at doses that achieved serum MPA levels comparable to those reported in clinical trials.
|
We conducted two independent in vivo metastasis experiments that differed slightly in the maintenance phase (Fig. 3, A). MDA-MB-231T cells (5 x 105) were injected intravenously into nude mice, which were then housed for 4 weeks to permit formation of micrometastases. A pathologist's examination of hematoxylin and eosinstained sections of lungs obtained from 4-week postinjection mice revealed micrometastases (i.e., clusters of tumor cells within the lung parenchyma) (Fig. 3, B). In Experiment 1, an induction dose of vehicle or 2 mg or 4 mg of MPA was then given twice weekly for the next 4 weeks; in Experiment 2 the 4-mg dose was dropped due to excessive weight gain, and mice were given MPA doses of 0.5, 1, and 2 mg. In Experiment 1, the 4 weeks of induction treatment was followed by a single maintenance injection of the induction dose given 4 weeks later. Large metastases developed in the mice within the maintenance period, and all mice in the experiment were killed 1 day after the maintenance dose was administered. In Experiment 2 the maintenance dose was modified to half the induction dose (0.25, 0.5, and 1 mg, respectively) every other week for 6 weeks to maintain serum MPA levels throughout the maintenance phase.
|
|
We tested the impact of increasing the MPA dose during the maintenance period in an experimental arm of Experiment 2 (data not shown). Mice were given 2 mg of MPA twice weekly throughout the study rather than twice weekly for 4 weeks and then once every other week, as was done in the induction/maintenance regimen. The mean numbers (range) of metastases were 15.8 (065) and 14.5 (055) for mice given 2 mg of MPA twice weekly and mice given 2 mg of MPA in the standard induction/maintenance regimen, respectively, and 33 (1110) in the vehicle control arm. There were no statistically significant differences in either the percentage of mice without metastases or the number of large metastases between the two treatment groups. Thus, increasing the frequency of MPA injections during the maintenance period did not further reduce metastatic colonization.
Analysis of Potential Side Effects of MPA Treatment
The reported human side effects of MPA include weight gain (4648) and decreased bone density (49). MPA induces mammary tumors in rodents, but only at doses 10-fold higher than those used in our experiments (50,51). In our study, all mice treated with MPA gained weight relative to vehicle-treated mice (Fig. 4). In Experiment 1, the 4-mg dose caused up to a 21% weight gain during the treatment period, compared with up to 30% for the 2-mg dose relative to the control (Fig. 4, A). In Experiment 2, mice were treated for a longer period. The 1- and 2-mg doses exerted graded effects on weight gain during the treatment period (7% and 16%, respectively, relative to 13% for the controls) (Fig. 4, B). Further examination of the weight gain was conducted by measuring the lean (muscle) and fat weight of 10 mice per treatment group from Experiment 2, using a dual energy x-ray absorptiometer (Table 3). The weight gain was balanced between lean and fat weight, and the distribution of weight gain (i.e., the balance of fat and lean) did not differ between MPA-treated and control mice. In addition to causing weight gain, steroids are known to affect bone density. The bones from 10 mice per treatment group in Experiment 2 were examined for bone mineral density and bone mineral content (Table 3). No statistically significant differences were observed between the control and the MPA treatment groups.
|
|
Nm23 Expression in Lung Metastases
To examine the effect of MPA treatment on Nm23-H1 expression in lung metastases, we sectioned the lungs of five metastasis-containing mice from each treatment group and subjected one randomly chosen section from each mouse to immunohistochemical staining for Nm23 (Fig. 5). Lung metastases were categorized as exhibiting homogeneous or heterogeneous high Nm23 expression or homogeneous low Nm23 expression based on the relative staining of metastases compared with that of surrounding lung parenchyma. All metastases in the stained section were scored. The metastases from the control mice exhibited a range of Nm23 expression intensities: 54% of lesions had homogeneous low expression, 13% had homogeneous high expression, and 33% had heterogeneous high expression. By contrast, mice treated with 1 or 2 mg MPA displayed homogeneous low Nm23 staining in only 9% and 6% of lesions, respectively, and homogeneous high Nm23 staining in 41% and 43% of lesions, respectively. In addition, the total percentages of metastases with homogeneous low expression were higher in control mice than in the 1 mg (P = .024) or 2 mg (P = .048) group. MPA at 0.5 mg had little or no stimulatory effect on Nm23 expression in metastases compared with vehicle alone.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our preclinical modeling effort also included an extensive pharmacokinetic evaluation designed to allow us to identify MPA doses and schedules that would lead to serum concentrations that are achievable in humans. Elevation of mammary fat pad Nm23-H1 expression in the ductal epithelial cells was used as a surrogate biologic endpoint for the pharmacokinetic study. However, factors such as serum binding proteins may differ between mice and humans, leading to differences in bioavailability even when serum concentrations appear to be similar.
The extent to which elevation of Nm23-H1 expression contributes to the phenotypic effects of MPA was estimated in colony formation assays. Antisense Nm23-H1 transfectants, which did not undergo the MPA-induced increase in Nm23-H1 expression, also exhibited no inhibition of colony formation. Although this result suggests that MPA exerts its suppressive effects on metastasis via its effect on Nm23-H1 expression, it is possible that MPA has additional suppressive activities in this model system. The in vitro and in vivo suppressive effects of MPA on breast neoplastic progression have been ascribed to other molecular events, mostly through the progesterone receptor (5464). In addition, MPA has been noted to exert stimulatory effects on breast neoplastic progression (65), particularly in combination with estrogen (66,67), and the riskbenefit ratio will determine the acceptability of this agent. Indeed, it was to avoid the effects of MPA on PR that we focused our preclinical experiments on ER/PR disease. Experiments are under way to define the microarray signature of elevated Nm23-H1 expression through gene transfection and MPA treatment to ascertain common signatures and divergent effects.
Our data suggest that MPA should be reevaluated, not as a PR-directed hormonal agent but rather as a GR-directed target for elevation of Nm23-H1 metastasis suppressor expression in aggressive, hormone receptornegative breast cancer. This compound would need to be delivered chronically, similar to the 1-month induction, monthly maintenance dosing used in previous clinical trials and in the mouse experiments reported here. We would not expect such an MPA dosing regimen to have any inhibitory effect on established metastatic disease, because the target of MPA is the outgrowth of micrometastases. Given the multiple known effects of MPA, only a molecularly defined subset of patients would be considered for a clinical trial: Postmenopausal patients with primary tumors that express low levels of Nm23-H1 and are also ER negative, PR negative, and GR positive. MPA may have distinct phenotypic effects in the context of a tumor cell that expresses PR (6870), which are untested herein. A dose-finding trial is under consideration in which breast cancer patients would receive MPA and the Nm23-H1 expression of a biopsiable metastasis would be determined to identify a biologically effective dose. A randomized trial of MPA therapy (after chemotherapy) using the molecularly defined cohort of high-risk patients defined above could then determine the effect of MPA on time to progression and survival.
![]() |
NOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Diane Palmieri and Douglas Halverson contributed equally to this report and should be considered joint first authors.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(1) Steeg P. Metastasis suppressors alter the signal transduction of cancer cells. Nat Cancer Rev 2003;3:5563.[CrossRef]
(2) Yoshida B, Sokoloff M, Welch D, Rinker-Schaeffer C. Metastasis-suppressor genes: a review and perspective on an emerging field. J Natl Cancer Inst 2000;92:171730.
(3) Shevde L, Welch D. Metastasis suppressor pathwaysan evolving paradigm. Cancer Lett 2003;198:120.[CrossRef][ISI][Medline]
(4) Steeg PS, Bevilacqua G, Kopper L, Thorgeirsson UP, Talmadge JE, Liotta LA, et al. Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst 1988;80:2004.[Abstract]
(5) Leone A, Flatow U, King CR, Sandeen MA, Margulies IM, Liotta LA, et al. Reduced tumor incidence, metastatic potential, and cytokine responsiveness of nm23-transfected melanoma cells. Cell 1991;65:2535.[CrossRef][ISI][Medline]
(6) Lacombe ML, Milon L, Munier A, Mehus J, Lambeth D. The human Nm23/nucleoside diphosphate kinases. J Bioenerg Biomembr 2000;32:24758.[CrossRef][ISI][Medline]
(7) Leone A, Flatow U, VanHoutte K, Steeg PS. Transfection of human nm23-H1 into the human MDA-MB-435 breast carcinoma cell line: Effects on tumor metastatic potential, colonization, and enzymatic activity. Oncogene 1993;8:232533.[ISI][Medline]
(8) Bhujwalla Z, Aboagye E, Gilles R, Chack V, Mendola C, Backer J. Nm23-transfected MDA-MB-435 human breast carcinoma cells form tumors with altered phospholipid metabolism and pH: a 31P nuclear magnetic resonance study in vivo and in vitro. Magnetic Res Med 1999;41:897903.[CrossRef][ISI]
(9) Russell R, Geisinger K, Mehta R, White W, Shelton B, Kute T. nm23Relationship to the metastatic potential of breast carcinoma cell lines, primary human xenografts and lymph node negative breast carcinoma patients. Cancer 1997;79:115865.[CrossRef][ISI][Medline]
(10) Fukuda M, Ishii A, Yasutomo Y, Shimada N, Ishikawa N, Hanai N, et al. Metastatic potential of rat mammary adenocarcinoma cells associated with decreased expression of nucleoside diphosphate kinase/nm23: reduction by transfection of NDP Kinase a isoform, an nm23-H2 gene homolog. Int J Cancer 1996;65:5317.[CrossRef][ISI][Medline]
(11) Baba H, Urano T, Okada K, Furukawa K, Nakayama E, Tanaka H, et al. Two isotypes of murine nm23/nucleoside diphosphate kinase, nm23-M1 and nm23-M2, are involved in metastatic suppression of a murine melanoma line. Cancer Res 1995;55:197781.[Abstract]
(12) Miele ME, Rosa AD, Lee JH, Hicks DJ, Dennis JU, Steeg PS, et al. Suppression of human melanoma metastasis following introduction of chromosome 6 is independent of NME1 (nm23). Clin Exp Metastasis 1997;15:25965.[CrossRef][ISI][Medline]
(13) Parhar RS, Shi Y, Zou M, Farid NR, Ernst P, Al-Sedairy S. Effects of cytokine mediated modulation of Nm23 expression on the invasion and metastatic behavior of B16F10 melanoma cells. Int J Cancer 1995;60:20410.[ISI][Medline]
(14) Tagashira H, Hamazaki K, Tanaka N, Gao C, Namba M. Reduced metastatic potential and c-myc overexpression of colon adenocarcinoma cells (Colon 26 line) transfected with nm23-R2 rat nucleoside diphosphate kinase a isoform. Int J Mol Med 1998;2:658.[ISI][Medline]
(15) Miyazaki H, Fukuda M, Ishijima Y, Negishi A, Hirayama R, Ishikawa N, et al. Overexpression of nm23-H2/NDP kinase B in a human oral squamous cell carcinoma cell line results in reduced metastasis, differentiated phenotype in the metastatic site, and growth factor-independent proliferative activity in culture. Clin Cancer Res 1999;5:43017.
(16) Suzuki E, Ota T, Tsukuda K, Okita A, Matsuoka K, Murakami M, et al. nm23-H1 reduces in vitro cell migration and the liver metastatic potential of colon cancer cells by regulating myosin light chain phosphorylation. Int J Cancer 2004;108:20711.[CrossRef][ISI][Medline]
(17) Kantor JD, McCormick B, Steeg PS, Zetter BR. Inhibition of cell motility after nm23 transfection of human and murine tumor cells. Cancer Res 1993;53:19713.[Abstract]
(18) Howlett AR, Petersen OW, Steeg PS, Bissell MJ. A novel function for Nm23: overexpression in human breast carcinoma cells leads to the formation of basement membrane and growth arrest. J Natl Cancer Inst 1994;86:183844.[Abstract]
(19) Russell R, Pedersen A, Kantor J, Geisinger K, Long R, Zbieranski N, et al. Relationship of nm23 to proteolytic factors, proliferation and motility in breast cancer tissues and cell lines. Br J Cancer 1998;78:7107.[ISI][Medline]
(20) Bemis L, Schedin P. Reproductive state of rat mammary gland stroma modulates human breast cancer cell migration and invasion. Cancer Res 2000;60:34148.
(21) Smukste I, Stockwell B. Restoring functions of tumor suppressors with small molecules. Cancer Cell 2003;4:41920.[CrossRef][ISI][Medline]
(22) Cropp C, Lidereau R, Leone A, Liscia D, Cappa A, Campbell G, et al. NME1 protein expression and loss of heterozygosity mutations in primary human breast tumors. J Natl Cancer Inst 1994;86:11679.[ISI][Medline]
(23) Hartsough M, Clare S, Mair M, Elkahloun A, Sgroi D, Osborne C, et al. Elevation of breast carcinoma nm23-H1 metastasis suppressor gene expression and reduced motility by DNA methylation inhibition. Cancer Res 2001;61:23207.
(24) Lin K, Wang W, Wu Y, Cheng S. Activation of antimetastatic Nm23-H1 gene expression by estrogen and its a-receptor. Endocrinology 2002;143:46775.
(25) Liu F, Qi HL, Chen HL. Effects of all-trans retinoic acid and epidermal growth factor on the expression of nm23-H1 in human hepatocarcinoma cells. J Cancer Res Clin Oncol 2000;126:8590.[ISI][Medline]
(26) Jiang W, Hiscox S, Bryce R, Horrobin D, Mansel R. The effects of n-6 polyunsaturated fatty acids on the expression of nm-23 in human cancer cells. Br J Cancer 1988;77:7318.
(27) Yu HG, Huang JA, Yang YN, Huang H, Luo HS, Yu JP, et al. The effects of acetylsalicylic acid on proliferation, apoptosis, and invasion of cyclooxygenase-2 negative colon cancer cells. Eur J Clin Invest 2002;32:83846.[CrossRef][ISI][Medline]
(28) Ouatas T, Clare S, Hartsough M, DeLaRosa A, Steeg P. MMTV-associated transcription factor binding sites increase nm23-H1 metastasis suppressor gene expression in human breast carcinoma cell lines. Clin Exp Metastasis 2002;19:3542.[CrossRef][ISI][Medline]
(29) Ouatas T, Halverson D, Steeg P. Dexamethasone and medroxyprogesterone acetate elevate Nm23-H1 metastasis suppressor expression in metastatic human breast carcinoma cells: new uses for old compounds. Clin Cancer Res 2003;9:376372.
(30) Skegg D, Noonan E, Paul C, Spears G, Meirik O, Thomas D. Depot medroxyprogesterone acetate and breast cancer. A pooled analysis of the World Health Organization and New Zealand studies. JAMA 1995;273:799804.[Abstract]
(31) Stockler M, Wilcken N, Ghersi D, Simes R. Systematic reviews of chemotherapy and endocrine therapy in metastatic breast cancer. Cancer Treat Rev 2000;26:15168.[CrossRef][ISI][Medline]
(32) Focan C, Beauduin M, Salamon E, Greve JD, deWasch G, Lobelle J, et al. Adjuvant high dose medroxyprogesterone acetate for early breast cancer: 13 years update in a multicentre randomized trial. Br J Cancer 2001;85:18.
(33) Hori T, Kodama H, Nishimura S, Hatano T, Okamura R, Fujii K, et al. A randomized study comparing oral and standard regimens for metastatic breast cancer. Oncol Rep 2001;8:106771.[ISI][Medline]
(34) Bryne M, Gebski V, Forbes J, Tattersall M, Simes R, Coates A, et al. Medroxyprogesterone acetate addition or substitution for tamoxifen in advanced tamoxifen-resistant breast cancer: A Phase III randomized trial. J Clin Oncol 1997;15:31418.[Abstract]
(35) Bentel J, Birrell S, Pickering M, Holds D, Horsfall D, Tilley W. Androgen receptor agonist activity of the synthetic progestin medroxyprogesterone acetate, in human breast cancer cells. Mol Cell Endocrinol 1999;154:1120.[CrossRef][ISI][Medline]
(36) Selman P, Wolfswinkel J, Mol J. Binding specificity of medroxyprogesterone acetate and proligestone for the progesterone and glucocorticoid receptor in the dog. Steroids 1996;61:1337.[CrossRef][ISI][Medline]
(37) Bamberger C, Else T, Bamberger A, Beil F, Shulte H. Dissociative glucocorticoid activity of medroxyprogesterone acetate in normal human lymphocytes. J Biol Chem 1999;84:405561.
(38) Lan L, Vinci J, Melendez J, Jeffrey J, Wilcox B. Progesterone mediates decreases in uterine smooth muscle cell interelukin-1a by a mechanism involving decreased stability of IL-1a mRNA. Mol Cell Endocrinol 1999;155:12333.[CrossRef][ISI][Medline]
(39) Shiozawa T, Horiuchi A, Kato K, Obinata M, Konishi I, Fuji S, et al. Up-regulation of p27Kip1 by progestins is involved in the growth suppression of the normal and malignant human endometrial glandular cells. Endocrinology 2001;142:41828.
(40) Hupperets P, Wils J, Volovics L, Schouten L, Fickers M, Bron H, et al. Adjuvant chemo-hormonal therapy with cyclophosphamide, doxorubicin and 5-fluroruacil (CAF) with or without medroxyprogesterone acetate (MPA) for node-positive cancer patients, update at 12 years follow up. The Breast 2001;10:357.[CrossRef][Medline]
(41) Nagy T, Clair A. Precision and accuracy of dual-energy X-ray absorptiometry for determining in vivo body composition of mice. Obes Res 2000;8:3928.
(42) Hochberg Y. A sharper Bonferroni procedure for multiple tests of significance. Biometrika 1988;75:8002.[ISI]
(43) Packard G, Boardman T. The misuse of ratios, indices, and percentages in ecophysiological research. Physiol Zool 1988;61:19.[ISI]
(44) Nishimura R, Nagao K, Matsuda M, Baba K, Matsuoka Y, Yamashita H, et al. Predictive value of serum medroxyprogesterone acetate concentration for response in advanced or recurrent breast cancer. Eur J Cancer 1997;33:140712.[CrossRef][Medline]
(45) Etienne M, Milano G, Frenay M, Renee N, Francois E, Thyss A, et al. Pharmacokinetics and pharmacodynamics of medroxyprogesterone acetate in advance breast cancer patients. J Clin Oncol 1992;10:117682.[Abstract]
(46) Davila E, Vogel C, East D, Cairns V, Hilsenbeck S. Clinical trial of high dose oral medroxyprogesterone acetate in the treatment of metastatic breast cancer and review of the literature. Cancer 1988;61:21617.[ISI][Medline]
(47) Gallagher CJ, Cairnduff F, Smith IE. High dose versus low dose medroxyprogesterone acetate: a randomized trial in advanced breast cancer. Eur J Cancer Clin Oncol 1987;23:1895900.[CrossRef][ISI][Medline]
(48) Koyama H, Tominaga T, Asaishi K, Abe R, Iino Y, Enomoto K, et al. A randomized controlled comparative study of oral medroxyprogesterone acetate 1,200 and 600 mg in patients with advanced or recurrent breast cancer. Oncology 1999;56:28390.[CrossRef][ISI][Medline]
(49) Ishida Y, Heersche J. Pharmacologic doses of medroxyprogesterone may cause bone loss through glucocorticoid activity: an hypothesis. Osteoporos Int 2002;13:6015.[CrossRef][ISI][Medline]
(50) Pazos P, Lanari C, Elizalde P, Montecchia F, Charreau E, Molinolo A. Promoter effect of medroxyprogesterone acetate (MPA) in N-methyl-N-nitrosourea (MNU) induced mammary tumors in BALB/c mice. Carcinogenesis 1998;19:52931.[Abstract]
(51) Lanari C, Molonolo A, Pasqualini C. Induction of mammary adenocarcinomas by medroxyprogesterone acetate in BALB/c mice. Cancer Lett 1986;33:21533.[CrossRef][ISI][Medline]
(52) Chekmareva M, Kadkhodaian M, Hollowell C, Kim H, Yoshida B, Luu H, et al. Chromosome 17-mediated dormancy of AT6.1 prostate cancer micrometastases. Cancer Res 1998;58:49639.[Abstract]
(53) Goldberg S, Harms J, Quon K, Welch D. Metastasis suppressed C8161 melanoma cells arrest in lung but fail to proliferate. Clin Exp Metastasis 1999;58:49639.
(54) Leo J, Guo C, Woon C, Aw S, Lin V. Glucocorticoid and mineralocorticoid cross-talk with progesterone receptor to induce focal adhesions and growth inhibition in breast cancer cells. Endocrinology 2004;145:131421.
(55) Lin V, Jin R, Tan PH, Aw SE, Woon CT, Bay BH. Progesterone induces cellular differentiation in MDA-MB-231 breast cancer cells transfected with progesterone receptor complementary DNA. Am J Pathol 2003;162:17817.
(56) Swarbrick A, Lee C, Sutherland R, Musgrove E. Cooperation of p27Kip1 and p18Ink4c in progestin-mediated cell cycle arrest in T-47D breast cancer cells. Mol Cell Biol 2000;20:258191.
(57) Alkhalaf M, El-Mowafy A. Overexpression of wild-type p53 gene renders MCF-7 breast cancer cells more sensitive to the antiproliferative effect of progesterone. J Endocrinol 2003;179:5562.
(58) Ahola T, Manninen T, Alkio N, Ylikomi T. G protein-coupled receptor 30 is critical for a progestin-induced growth inhibition in MCF-7 breast cancer cells. Endocrinology 2002;143:337684.
(59) Sugimoto T, Shiba E, Watanabe T, Takai S. Suppression of parathyroid hormone-related protein messenger RNA expression by medroxyprogesterone acetate in breast cancer tissues. Breast Cancer Res Treat 1999;56:1123.[CrossRef][ISI][Medline]
(60) Thuneke I, Shulte H, Bamberger A. Biphasic effect of medroxyprogesterone acetate (MPA) treatment on proliferation and cyclin D1 gene transcription in T47D breast cancer cells. Breast Cancer Res Treat 2000;63:2438.[CrossRef][ISI][Medline]
(61) Mizukami Y, Tajiri K, Nonomura A, Nogichi M, Taniya T, Koyasaki N, et al. Effects of tamoxifen, medroxyprogesterone acetate and estradiol on tumor growth and oncogene expression in MCF-7 breast cancer cell line transplanted into nude mice. Anticancer Res 1991;11:13338.[ISI][Medline]
(62) Yamaji T, Tsuboi H, Murata N, Uchida M, Kohno T, Sugino E, et al. Anti-angiogenic activity of a novel synthetic agent, 9-a-flurormedroxyprogesterone acetate. Cancer Lett 1999;145:10714.[CrossRef][ISI][Medline]
(63) Yuyama Y, Yagihashi A, Hirata K, Suzuki Y, Ohmura T, Okazaki M, et al. The effects of epirubicin hydrochloride (EPI) plus pretreatment of medroxyprogesterone acetate (MPA) on FM3A breast cancer cells transplanted in female C3H/He mice. In Vivo 2003;17:2514.[ISI][Medline]
(64) vanVeelen H, Willemse P, Sleijfer D, Sluiter W, Doorenbos H. Endocrine effects of medroxyprogesterone acetate: relation between plasma levels and suppression of adrenal steroids in patients with breast cancer. Cancer Treat Rep 1985;9:97783.
(65) Sunil N, Bennett J, Haslam S. Hepatocyte growth factor is required for progestin-induced epithelial cell proliferation and alveolar-like morphogenesis in serum-free culture of normal mammary epithelial cells. Endocrinology 2002;143:295360.
(66) Ballare C, Uhrig M, Bechtold T, Sancho E, DiDomenico M, Migliaccio A, et al. Two domains of the progesterone receptor interact with the estrogen receptor and are required for progesterone activation of the c-Src/Erk pathway in mammalian cells. Mol Cell Biol 2003;23:19942008.
(67) Seeger H, Wallwiener D, Mueck A. The effect of progesterone and synthetic progestins on serum- and estradiol-stimulated proliferation of human breast cancer cells. Horm Metab Res 2003;35:7680.[CrossRef][ISI][Medline]
(68) Wan Y, Nordeen S. Overlapping but distinct gene regulation profiles by glucocorticoids and progestins in human breast cancer cells. Mol Endocrinol 2002;16:120414.
(69) Deroo B, Archer T. Differential activation of the IkBa and mouse mammary tumor virus promoters by progesterone and glucocorticoid receptors. J Steroid Biochem Mol Biol 2002;81:30917.[CrossRef][ISI][Medline]
(70) Song LN, Huse B, Rusconi Simons SS Jr. Transactivation specificity of glucocorticoid versus progesterone receptors. J Biol Chem 2001;276:2480616.
Manuscript received October 22, 2004; revised February 28, 2005; accepted March 2, 2005.
This article has been cited by other articles in HighWire Press-hosted journals:
Correspondence about this Article
Editorial about this Article
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |