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Clinical Cancer Research 13, 2751, May 1, 2007. doi: 10.1158/1078-0432.CCR-06-2466
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway Improves Response of Long-term Estrogen-Deprived Breast Cancer Xenografts to Antiestrogens

Gauri Sabnis1, Olga Goloubeva2, Danijela Jelovac1, Adam Schayowitz1 and Angela Brodie1,2

Authors' Affiliations: 1 Department of Pharmacology and Experimental Therapeutics, University of Maryland Baltimore and 2 University of Maryland Greenebaum Cancer Center, Baltimore, Maryland

Requests for reprints: Angela Brodie, Department of Pharmacology and Experimental Therapeutics, University of Maryland Baltimore, School of Medicine, Health Science Facility I, Room 580G, 685 West Baltimore Street, Baltimore, MD 21201. Phone: 410-706-3137; Fax: 410-706-0032; E-mail: abrodie{at}umaryland.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Aromatase inhibitors that block the synthesis of estrogen are proving to be superior to antiestrogens and may replace tamoxifen as first-line treatment for postmenopausal estrogen receptor (ER)–positive breast cancer patients. However, acquisition of resistance to all forms of treatments is inevitable and a major clinical concern. In this study, we have investigated the effects of long-term estrogen deprivation in the breast cancer xenograft model and whether sensitivity to antiestrogens can be restored in vivo. We also compared whether combining wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone.

Experimental Design: Long-term estrogen-deprived aromatase-transfected human ER-positive breast cancer cells (UMB-1Ca) were grown as tumors in ovariectomized athymic nude mice. Twelve weeks after inoculation, when tumors reached 300 mm3, animals were grouped and injected with vehicle, {Delta}4A, letrozole, tamoxifen, fulvestrant, wortmannin, tamoxifen plus wortmannin, and wortmannin plus fulvestrant. Tumor volumes were measured weekly.

Results: Tumors of UMB-1Ca cells grew equally well with and without androstenedione, indicating the ability of the cells to proliferate in the absence of estrogen. The combination of wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone. The combination of wortmannin plus fulvestrant was the most effective treatment that maintained tumor regression for a prolonged time.

Conclusion: These results suggest that blocking both ER and growth factor receptor pathways could provide effective control over tumor growth of long-term estrogen-deprived human breast cancers.

Estrogen is the major stimulus to breast cancer progression in both premenopausal and postmenopausal patients. In young women, the main source of estrogen is the ovary. After menopause, ovarian production declines and extragonadal sites such as adipose tissue are the main source of circulating estrogen (1). However, tissue concentrations within the breast are comparable with those of premenopausal women (2, 3). The actions of estrogen on the tumor are mediated by estrogen receptor (ER). In breast cancer patients, tumor ER concentrations are higher after menopause, resulting in cancers that are sensitive to even low levels of circulating estrogens (4). A large proportion of breast cancer patients are postmenopausal women with ER-positive tumors, and treatment strategies that inhibit the growth stimulus of estrogen are effective in providing long-term benefit. Aromatase inhibitors that reduce estrogen production have now been shown to be effective treatment for breast cancer (5, 6). The triazole compound letrozole has been found to be superior to tamoxifen (7, 8). Anastrozole, a similar triazole compound, was more effective than tamoxifen in ER-positive patients and had few side effects (9). These clinical findings are consistent with results from previous investigations in our tumor model (10–12). The model mimics the postmenopausal breast cancer patient because the source of estrogen is from nonovarian tissue and is not under gonadotropin regulation (13). However, because the mouse has no significant production of peripherally formed estrogen, the MCF-7 human breast cancer cells stably transfected with the human aromatase gene serve as an autocrine source of estrogen to stimulate tumor growth in the ovariectomized, immunosuppressed mice. The resulting tumors synthesize estrogen from supplemented androstenedione ({Delta}4A) and proliferate in response to estrogen acting via the ER. These tumors are therefore useful for studying the effects of both antiestrogens and aromatase inhibitors in the same model system (13, 14). This model has provided valuable data that have predicted the efficacy of aromatase inhibitors in breast cancer patients. We have reported a number of studies on growth effects of aromatase inhibitors and antiestrogens used in different combinations and sequences in the MCF-7Ca xenograft model (15, 16). These studies clearly showed the superiority of aromatase inhibitors over antiestrogen tamoxifen, and these findings have now been confirmed by numerous clinical trails (17, 18). Although tumor growth was suppressed over an extended period by aromatase inhibitors compared with tamoxifen, eventually tumors started to regrow even in the presence of aromatase inhibitors. To investigate the mechanisms in the loss of sensitivity of the tumors to aromatase inhibitors, we developed an in vitro model system in which MCF-7Ca cells are deprived of estrogens in culture, designated as UMB-1Ca cells (16, 19). Similar to other studies of long-term estrogen deprived MCF-7 cells (20), alternative signaling pathways were activated in UMB-1Ca cells that enabled them to proliferate in the absence of E2. UMB-1Ca cells showed increased activation of Akt in addition to downstream antiapoptotic and proliferative proteins. Our previous results have shown cross-talk between the ER{alpha} and Akt pathways in these cells and suggest that blockade of both of these pathways could more effectively regulate cancer cell proliferation (19). To provide a more physiologically relevant model, we did an in vivo study using the UMB-1Ca cells inoculated in ovariectomized female nude mice. On tumor formation, mice were treated with phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin alone or in combination with antiestrogen tamoxifen or fulvestrant to determine the effect on tumor growth (21, 22).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials. Improved MEM, penicillin/streptomycin solution (10,000 IU each), 0.25% trypsin-1 mmol/L EDTA solution, Dulbecco's PBS, and geneticin (G418) were obtained from Invitrogen (Carlsbad, CA). Fetal bovine serum was obtained from Hyclone (Logan, UT). PI3K inhibitor wortmannin, androstenedione, tamoxifen, Matrigel, and Tween 20 were obtained from Sigma Chemical Co. (St. Louis, MO). Antibodies against p-Akt (Ser473 and Thr308), p-PDK-1, Akt, and progesterone receptor were purchased from Cell Signaling Technology (Beverly, MA).

MCF-7 human breast cancer cells stably transfected with the human aromatase gene (MCF-7Ca) were provided by Dr. S. Chen (City of Hope, Duarte, CA; ref. 13). Letrozole (Femara, CGS 20267) was provided by Dr. D. Evans (Novartis Pharma, Basel, Switzerland). The pure antiestrogen fulvestrant (Faslodex, ICI 182,780) was supplied by Dr. A. Wakeling (AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom). ECL chemiluminiscence kit and Hybond-ECL nitrocellulose membranes were purchased from Amersham Biosciences (Piscataway, NJ).

Tumor growth in ovariectomized female athymic nude mice. All animal studies were done according to the guidelines and approval of the Animal Care Committee of the University of Maryland School of Medicine. Female ovariectomized BALB/c athymic nude mice, 4 to 6 weeks of age, were obtained from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD). The mice were housed in a pathogen-free environment under controlled conditions of light and humidity and received food and water ad libitum.

UMB-1Ca cells were developed from MCF-7Ca cells as described earlier by culturing the cells in steroid-depleted medium, which consisted of phenol red–free improved MEM supplemented with 5% dextran-coated charcoal-treated serum, 1% penicillin/streptomycin, and 750 µg/mL G418. The tumor xenografts were grown in mice as previously described. Subconfluent cells were scraped into Dulbecco's PBS, collected by centrifugation, and resuspended in Matrigel (10 mg/mL) at 2.5 x 107/mL. Each mouse received s.c. inoculations in one site per flank with 100 µL of cell suspension. Mice in one group (n = 10) were then injected daily with {Delta}4A (100 µg/d) in the vehicle (0.3% hydroxy propyl cellulose in 0.9% NaCl) for the duration of the experiment. Mice in a second group (n = 10) were then injected daily with {Delta}4A (100 µg/d) plus letrozole (10 µg/d) for the duration of the experiment, and the remaining mice were injected with vehicle alone. Measurements and treatments began when the tumors reached a measurable size (~300 mm3), ~12 weeks after cell inoculation. Mice were assigned to groups for treatment so that there was no statistically significant difference in tumor volume among the groups at the beginning of the treatment. Tumors were measured twice weekly with calipers and volumes were calculated using the formula (4/3) {pi} r12 r2 (r1 ≤ r2). All drugs were prepared in 0.3% hydroxy propyl cellulose (except wortmannin). Wortmannin for injection was prepared as 50 mg/mL stock solution in DMSO and then diluted in 0.9% NaCl solution to obtain the required concentration. The animals that were injected with vehicle alone were then treated for the indicated times. Mice were then injected s.c. daily with the indicated drugs: 100 µg/mouse/d (5x weekly) of {Delta}4A plus 10 µg/mouse/d (5x weekly) of letrozole or 100 µg/mouse/d (5x weekly) of tamoxifen and 1,000 µg/mouse/d (5x weekly) of fulvestrant. The doses of letrozole, {Delta}4A, tamoxifen, or fulvestrant used are as previously determined and reported (15, 16). Mice in the wortmannin group received 350 µg/kg/d of the drug i.p. twice weekly. Mice in the {Delta}4A and {Delta}4A plus letrozole groups were treated for 10.5 weeks, during which time tumors had grown >500% of the initial volume. Mice in the control, tamoxifen, and wortmannin groups were treated for 14 weeks. Because the tumors had grown >600% of the initial volume, they were sacrificed by decapitation and the trunk blood was collected. Mice in the fulvestrant, tamoxifen plus wortmannin, and fulvestrant plus wortmannin groups were treated for 17 weeks, after which they were sacrificed by decapitation and the trunk blood was collected. Tumors and uteri were excised, cleaned, weighed, and stored at –80°C for additional analysis.

Western blotting. The protein extracts from tumor tissues were prepared by homogenizing the tissue in ice-cold Dulbecco's PBS containing protease inhibitors. Equal amounts (50 µg) of protein from each sample were separated on a denaturing polyacrylamide gel and transferred to nitrocellulose membrane. The protein-bound membranes were then incubated for 1 h at room temperature with 0.1% Tween 20 in TBS and 5% nonfat dry milk to block nonspecific antibody binding. The membranes were then incubated with respective primary antibodies as specified in manufacturer's protocol, and specific binding was visualized by using species-specific immunoglobulin G followed by enhanced chemiluminiscent detection and exposure to ECL X-ray film. Bands were quantitated by densitometry using Molecular Dynamics Software (ImageQuant, Sunnyvale, CA). The densitometric values are corrected for loading control shown in the bottom.

In vitro protein kinase assay. The Akt kinase assay was carried out according to the manufacturer's protocol. Briefly, Akt protein in 200-µL tumor lysates, which contain ~200 µg total proteins, was immunoprecipitated with immobilized monoclonal antibody for Akt (Cell Signaling Technology). The next day, the reaction was carried out in 50 µL of 1x kinase buffer supplemented with 200 µmol/L ATP and 2 µg of glycogen synthase kinase-3{alpha}/ß fusion protein and incubated for 30 min at 30°C. The reaction is terminated by addition of 25-µL 3x SDS sample buffer. The samples were boiled for 5 min, vortexed, and then microcentrifuged for 2 min. The sample (30 µL) was then loaded on to a SDS-PAGE gel and analyzed by Western blotting. The gel was probed with phospho antibody for glycogen synthase kinase-3{alpha}/ß. The bands were quantitated using ImageQuant (Molecular Dynamics).

Statistics. Data on tumor volume and weight, as well as uterine weight, were analyzed separately. Growth curve models were used to summarize the pattern of response over treatment duration. For tumor growth, diagnostic plots suggested that the model of exponential growth was appropriate to the data. Therefore, tumor volumes were transformed to ln(volume + 1), adding 1 to all volume measurements, including zeros. We used linear mixed-effects models to estimate an exponential variable controlling the tumor growth rate for each of the six treatment groups. The tumor growth rate (the slope of the regression line) was compared between prespecified treatment groups. Responses from different animals were assumed to be statistically independent whereas those within a subject were correlated. The mean tumor volume was modeled as a combination of fixed effects shared by all subjects and random effects unique to a particular animal. We used random effects for both intercept and slope at animal level. In the longitudinal assessment of tumor growth, we have chosen not to assume any specific form for the random effects covariance matrix. The simple covariance structure was selected for measurement within a mouse. The baseline or start of treatment tumor volume was included as a covariate in the resulting regression model. The results of treatment group comparisons are presented as differences in exponential variables controlling the growth rate with the corresponding 95% confidence intervals. For the analyses of uterine weight data, we used logarithmic transformation to stabilize variance. General linear models were used for the uterine data. The proportions of tumors that shrunk below a threshold during treatment were compared using Fisher's exact test. The treatment groups were compared with one another at 0.05 level of statistical significance. All reported P values were two sided. Mixed-effects models were used to compare average tumor volume on prespecified time points. The Tukey-Kramer method was used to adjust for multiple comparisons.

For in vitro studies, comparison of all treatment was made with vehicle-treated control using one-way ANOVA (Tukey's Test) on Sigma Plot 9.0 software (Point Richmond, CA). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Growth response of UMB-1Ca cells to treatment with {Delta}4A and {Delta}4A plus letrozole in vivo. Long-term estrogen-deprived UMB-1Ca cells were developed as reported earlier (16). Briefly, low-passage MCF-7Ca cells were cultured in steroid-depleted medium for 6 to 8 months. After a brief period of quiescence, the growth of the cells resumed and the cell line was designated as UMB-1Ca. These cells were insensitive to the effect of {Delta}4A or E2 and significantly less sensitive to letrozole in vitro, whereas in parental MCF-7Ca cells, proliferation was increased after treatment with {Delta}4A or E2 and markedly inhibited by treatment with letrozole (19).

Furthermore, when UMB-1Ca cells were inoculated into groups of ovariectomized nude mice and injected s.c. daily with either vehicle, {Delta}4A (100 µg/d), or {Delta}4A plus aromatase inhibitor letrozole (10 µg/d), tumors grew equally well in the presence and absence of {Delta}4A (Fig. 1A ), suggesting that the tumors no longer require estrogens for growth. This finding is consistent with our previous report (16, 19) that UMB-1Ca cells have adapted to grow in estrogen-deprived medium and did not require the aromatase substrate {Delta}4A for proliferation, regardless of high aromatase activity (16). In addition, UMB-1Ca xenografts also grew equally well in the presence and absence of letrozole (10 µg/d), indicating loss of sensitivity of the tumors to aromatase inhibitor therapy with letrozole (Fig. 1A). In week 10 of treatment, these mice were sacrificed due to large tumor size. The mean tumor weight was found to be 936.5 ± 232.2 mg in {Delta}4A group and was not statistically different from the mean tumor weight of {Delta}4A plus letrozole group, 867.0 ± 143.0 mg (Fig. 2A). The mean uterine weight of vehicle-treated mice was 18.8 ± 1.3 mg. The mean uterine weight of mice receiving {Delta}4A was 38.9 ± 1.8 mg (Fig. 2B ). This suggests that aromatase activity in the UMB-1Ca cells was sufficient to produce enough estrogen from {Delta}4A to maintain the uterine weight (16). The mean uterine weight in the letrozole-treated group was 11.0 ± 1.4 mg, significantly less than in the control and {Delta}4A treated groups (P < 0.001), suggesting that estrogen synthesis is inhibited by letrozole in these mice compared with vehicle-treated mice. The low uterine weight suggests that letrozole effectively inhibited the conversion of {Delta}4A to estrogens although the suppression of estrogen did not result in tumor growth inhibition.


Figure 1
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  		Fig. 1. A, effects of {Delta}4A and {Delta}4A plus letrozole on the growth of UMB-1Ca xenografts. Each mouse received s.c. injections at one site on each flank with 100 µL of suspension of UMB-1Ca cells (2.5 x 107/mL). Mice were divided into three groups (n = 10) and injected s.c. daily with vehicle (n = 7), {Delta}4A 100 µg/d (n = 10), or {Delta}4A plus letrozole 10 µg/d (n = 10) from the day of inoculation. Measurements began when the tumors reached a measurable size (~300 mm3) and tumor volumes were measured twice weekly. B, effects of tamoxifen and fulvestrant alone or in combination with wortmannin on the growth of UMB-1Ca xenografts. Measurements and treatments began when the tumors reached a measurable size (~300 mm3) and tumor volumes were measured twice. The animals that were injected with vehicle alone were then treated for the indicated times. Mice were assigned to groups for treatment so that there was no statistically significant difference in tumor volume among the groups at the beginning of the treatment. Mice were then injected s.c. daily with the indicated drugs: 100 µg/mouse/d (5x weekly) of tamoxifen and 1 mg/mouse/d (5x weekly) of fulvestrant. Mice in wortmannin group received 0.35 mg/kg/d i.p. 2x weekly of the drug. The mice in control group received vehicle alone.

 

Figure 2
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  		Fig. 2. The effect of tamoxifen (10 µg/d) and fulvestrant (1 mg/d) alone or in combination with wortmannin (0.35 mg/kg/d) on tumor weight (A) and uterine weight (B) in female ovariectomized athymic nude mice bearing UMB-1Ca breast tumors. After the tumors reached a measurable size, animals were assigned to four groups with similar tumor volumes and injected s.c. daily with vehicle (control; n = 7), fulvestrant (1 mg/d; n = 7), tamoxifen (100 µg/d; n = 7), wortmannin (0.35 mg/kg/d), and the combination of tamoxifen (100 µg/d) plus wortmannin (0.35 mg/kg/d; n = 7) or the combination of fulvestrant (1 mg/d) plus wortmannin (0.35 mg/kg/d; n = 7). Mice in one group (n = 10) received {Delta}4A 100 µg/d and in one group (n = 10) received {Delta}4A plus letrozole (10 µg/d). The plot shows tumor weights (A) and uterine weights (B) of mice from the indicated treatment groups. *, P < 0.001, compared with all treatment groups; **, P < 0.001, compared with vehicle-treated control.

 
Treatment of UMB-1Ca xenografts with PI3K inhibitor wortmannin alone or in combination with antiestrogen tamoxifen or fulvestrant. After ~12 weeks, mice were randomized in various treatment groups (week 0). The mean tumor volume is plotted in Fig. 1B. Tumor volume doubled in vehicle-treated controls in 2 weeks and showed a 10-fold increase in volume by 14 weeks. These mice were sacrificed at this time due to large tumor size. The mean tumor weight was found to be 977.8 ± 180.0 mg.

Tumor growth was not inhibited in UMB-1Ca xenografts by tamoxifen (100 µg/d) treatment. The mean tumor volume doubled at 1.5 weeks and these mice were also sacrificed after 14 weeks (Fig. 1B). As shown in Fig. 2A and B, the mean tumor weight was 810.0 ± 93.1 mg and mean uterine weight was 73 ± 3.3 mg. The high uterine weight in the tamoxifen-treated group is consistent with the estrogenic effects of tamoxifen on the uterus as previously reported (16). Tumors of mice treated with tamoxifen continued to proliferate and had a similar estimated growth rate (exponential variable ß = 0.036 ± 0.007) as tumors of mice in the control group (ß = 0.027 ± 0.007).

Wortmannin (350 µg/kg/d) was not a potent inhibitor of tumor growth. The tumor growth rate (ß = 0.019 ± 0.007) was similar to vehicle-treated mice (P = 0.23). However, wortmannin-treated group had a significantly lower mean tumor volume than tamoxifen-treated group (P = 0.0004) in week 4. Tumor volume doubled in 5.5 weeks and increased 7-fold by week 14. At this time, these mice were sacrificed and tumors and uteri were collected and stored for analysis. The mean tumor weight of wortmannin-treated mice was 782.5 ± 231.5 mg. The mean uterine weight was 23.1 ± 4.4 mg, which was not significantly different from the control group. This suggests that wortmannin lacks any estrogenic or antiestrogenic properties.

The combination of wortmannin plus tamoxifen was found to be better than single agent tamoxifen (P = 0.04), showing that wortmannin was, to some extent, able to restore the growth inhibitory effects of tamoxifen (Fig. 1B). Regardless, the effect of this combination was of limited duration because the mean tumor volumes increased 2-fold in 2.5 weeks. However, tumors were static from week 3 through week 11. The addition of wortmannin to tamoxifen prolonged the time to progression and suggests partial restoration of responsiveness of UMB-1Ca xenografts to tamoxifen therapy.

Fulvestrant (1,000 µg/d) alone did not cause any significant regression of the tumors; however, growth was static until week 9 (ß = –0.0004 ± 0.006), at which point growth resumed and final tumor volume was increased 3-fold at termination of the experiment. The mean tumor weight at this time was 518.7 ± 145.7 mg and mean uterine weight was 6.0 ± 0.8 mg. Lower uterine weight compared with the vehicle control group suggests that there is residual estrogen in the ovariectomized mice, which is antagonized by fulvestrant. Fulvestrant was considerably more effective than tamoxifen in suppressing tumor growth. The mean tumor weight at the termination was significantly lower (P < 0.001) than all the other groups, although this group was sacrificed at the latest time point.

The combination of fulvestrant plus wortmannin treatment showed a synergistic effect and tumors responded for a longer duration. Before termination, tumor volume had reached the initial size. Importantly, the mean tumor volume in the wortmannin plus fulvestrant group was significantly lower than with wortmannin (P < 0.0001) or fulvestrant alone (P < 0.0023) at weeks 4, 10, 14, and 17. When compared with the wortmannin plus fulvestrant combination, differences in exponential variable governing growth rate (with the corresponding 95% confidence intervals) were 0.051 (0.032-0.070) for wortmannin and 0.031 (0.012-0.050) for fulvestrant. The proportion of tumors that have decreased in volume during treatment was 78.6% (11 of 14), 42.8% (6 of 14), and 7.1% (1 of 14) in fulvestrant plus wortmannin, fulvestrant, and wortmannin groups, respectively. There is sufficient evidence to conclude that the treatments affect the proportion of tumors that regressed (exact P = 0.0007). Adjusting for multiple comparisons, the two groups, wortmannin and wortmannin plus fulvestrant, are also different with regard to tumor regression; the two-sided P value was <0.001. These results are consistent with the in vitro growth inhibitory effects previously observed (19). The combination of fulvestrant plus wortmannin was found to be significantly better than all treatment groups and control at weeks 4, 10, 14, and 17 (P < 0.0001).

Modulation in the expression of ER{alpha} and proteins in the PI3K/Akt pathway after treatment with wortmannin and/or antiestrogens. At the end of the tumor growth study, the mice were sacrificed and tumors and uteri excised and weighed for further analysis of protein expression. The tumor lysates were examined for p-Akt (Ser473 and Thr308) expression (Fig. 3 ). The tumors treated with wortmannin alone or in combination with tamoxifen or fulvestrant showed reduction in the expression of p-Akt (densitometric values are corrected for loading control with expression of total Akt; Fig. 3). Significantly, fulvestrant alone was also found to be effective in reducing p-Akt (Ser473 and Thr308) expression. However, p-PDK-1 (upstream of Akt) was not affected by fulvestrant treatment (Fig. 3C). This suggests activation of Akt may not only occur through PI3K activation, but could be through ER, further implicating cross-talk between the two signaling pathways. However, tamoxifen alone did not affect the expression of any of these proteins. The inability of tamoxifen in inhibiting the cross-talk between the PI3K and ER{alpha} pathways may be due to the partial agonistic effects of tamoxifen or to the agonistic action of tamoxifen in cells with high AIB-1 levels.3


Figure 3
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  		Fig. 3. Western immunoblotting analysis of tumor lysates from UMB-1Ca xenografts. Blot shows phospho-Akt (Ser473) and phospho-Akt (Thr308) at 60 kDa, p-PDK-1 (Ser241) at 58 to 62 kDa, total-Akt at 60 kDa, ER{alpha} at 66 kDa, progesterone receptor-A and progesterone receptor-B (PgR) at 90 and 118 kDa, and ß-actin at 45 kDa. Experimental protocol was as described in Materials and Methods. Lane 1, control; lane 2, tamoxifen (100 µg/d); lane 3, wortmannin (0.35 mg/kg/d); lane 4, fulvestrant (1 mg/d); lane 5, wortmannin plus tamoxifen; and lane 6, wortmannin plus fulvestrant. Blots were stripped and probed for ß-actin to verify equal amount of protein loaded in each lane. Representative of three independent experiments; densitometric measurements are corrected for ß-actin.

 
The tumor lysates were examined for ER{alpha} expression. ER{alpha} protein expression was slightly reduced in wortmannin-treated tumors (Fig. 3). Fulvestrant, known as a selective ER down-regulator, was more effective than wortmannin in reducing the ER{alpha} levels in UMB-1Ca xenografts. Treatment of UMB-1Ca xenografts with wortmannin showed a slight reduction in the expression of progesterone receptor, suggesting little inhibition of ER{alpha}-mediated transcription. However, treatment with fulvestrant alone or wortmannin plus fulvestrant showed ~50% reduction in progesterone receptor levels (Fig. 3), suggesting inhibition of ER{alpha}-mediated transcription by fulvestrant but not by wortmannin.

When Akt kinase activity was measured in these tumor lysates, all the treatments, except tamoxifen alone, were found to inhibit the kinase activity to some extent compared with control samples (Fig. 4A ). The combination of fulvestrant with wortmannin was most effective in reducing Akt kinase activity. Furthermore, interaction between AKT and ER{alpha} was examined when tumor lysates from UMB-1Ca xenografts were first immunoprecipitated with anti-AKT antibody and then immunoblotted with the antibody for ER{alpha}. UMB-1Ca xenografts treated with the combination of fulvestrant plus wortmannin significantly (P < 0.01) inhibited this association compared with other treatment groups (Fig. 4B). This finding again correlates with the significant tumor growth inhibition. Similarly, single agent fulvestrant was moderately effective in inhibiting the association and tumor growth. Additionally, as shown in Fig. 4A, fulvestrant decreased ER{alpha} expression. However, the combination of fulvestrant plus wortmannin reduced ER{alpha} expression and the association to a much greater extent, the effect being significantly different from that of fulvestrant alone. Thus, the complete down-regulation of this association in wortmannin plus fulvestrant treatment group indicates the necessity of deactivation or down-regulation of both Akt and ER{alpha}. Thus, it is clear from the study that inhibition of both pathways and the cross-talk between the two pathways are essential for better control over tumor growth.


Figure 4
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  		Fig. 4. A, Akt kinase assay of tumor lysates from UMB-1Ca xenografts. Two-hundred microliters of tumor cell lysates containing ~200 µg of proteins were subjected to immunoprecipitation with immobilized anti-Akt antibody. The next day, after adding kinase reaction buffer and glutathione S-transferase fusion protein of glycogen synthase kinase-3{alpha}/ß (p-GSK3{alpha}/ß; Akt kinase substrate), samples were incubated at 30°C for 30 min. The reaction was terminated by addition of 3x sample buffer and samples were analyzed by Western blotting as described in Materials and Methods. Representative of three independent experiments. B, coimmunoprecipitation of tumor lysates from UMB-1Ca xenografts to examine association between ER{alpha} and Akt. Coimmunoprecipitation tumor lysates from UMB-1Ca xenografts for ER{alpha} protein and Akt. The experimental protocol was as described in Materials and Methods. Two-hundred microliters of tumor lysates were subjected to immunoprecipitation with anti-Akt antibody and then were subjected to Western immunoblotting with ER{alpha} antibody. Representative of three independent experiments. Blots were stripped and probed for Akt to verify equal amount of protein immunoprecipitated in each lane.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
There has been significant improvement in the efficacy of hormone therapy of breast cancer after the introduction of aromatase inhibitors (18, 23). Despite reduced estrogen production in postmenopausal breast cancer patients, a major clinical concern is the inevitable acquisition of resistance to treatment as the cancer cells adapt to the treatment environment. Our recent in vitro studies involving the long-term estrogen-deprived aromatase-transfected MCF-7 cells (UMB-1Ca; ref. 19) have shown activation of growth factor receptor signaling cascades that lead to growth stimulation and loss of responsiveness to endocrine therapy involving aromatase inhibitors (19). The key growth factor receptor pathways that regulate the cell cycle and transcription of genes involved in proliferation include Her-2 and the downstream kinase cascade. In the UMB-1Ca estrogen-deprived cells, PI3K and Akt are up-regulated compared with parental MCF-7Ca cells. ER also seems to be crucial in these resistant cells as cross-talk between ER and the signal transduction pathways seems to be responsible for the ability of the cells to adapt to a low-estrogen environment (19) and grow in the presence of aromatase inhibitors (24). Our experimental data suggest that both fulvestrant, which reduces ER levels, and a signal transduction inhibitor that blocks the PI3K/Akt pathway in these cells can inhibit tumor growth more effectively compared with each treatment alone (21).

This article focuses on the synergistic effect of combining an inhibitor of the PI3K/Akt pathway, such as wortmannin, with fulvestrant on tumor growth inhibition in breast cancer cells deprived of estrogen for an extended period of time. The pure antiestrogen, fulvestrant, was able to maintain tumor regression at 9 weeks (21). As expected, ER{alpha} expression in the tumors treated for 17 weeks was significantly reduced after fulvestrant treatment. Although wortmannin alone was not effective in suppressing tumor growth, wortmannin treatment was found to better (P = 0.0004) than tamoxifen till week 10. The addition of wortmannin to the tamoxifen regimen was found to prolong the responsiveness of tumors, shifting the growth curve to the right. The combination of fulvestrant plus wortmannin was significantly more effective than any other treatment because tumor growth was inhibited for a prolonged period (>10 weeks). Furthermore, at the end of the experiment, the tumor volume was less than twice the initial volume. In animals treated with fulvestrant plus wortmannin, tumors had a lower growth rate than animals treated with either wortmannin or animals that received fulvestrant. Tumors from the combined treatment group expressed both ER{alpha} and p-Akt at a very low level. The Akt kinase activity was lowest and the association between ER{alpha} and Akt was also significantly reduced. Although wortmannin is not a specific PI3K inhibitor and not used clinically, our studies show the importance of inhibiting the PI3K/Akt pathway in overcoming resistance to antiestrogen treatment. It has been suggested that an increase in growth factor and cellular kinase signaling in breast cancer potentates the ER pathway, which in turn reactivates growth factor signaling via genomic and nongenomic actions. This is believed to result in a stimulatory cycle that intensifies the activities of both ER and growth factor receptors (25).

Our results show that ER signaling can also be regulated by the complex growth factor signaling pathways. Bidirectional cross-talk is evident in many in vitro and in vivo model systems as well as some breast cancer patients (26). Our results suggest that tumor cells adapt to estrogen deprivation by up-regulating the estrogen signaling pathway and activating kinase signaling proteins to maintain transcription and cell proliferation. The involvement of ER{alpha} is supported by inhibition of growth of hormone refractory UMB-1Ca xenografts sensitive to fulvestrant. The combination of wortmannin plus fulvestrant had a significantly lower growth rate and also showed more pronounced inhibition of kinase signaling and ER{alpha}-mediated transcription than either compound alone. Thus, molecular communications from several intracellular kinases can alter the function of ER and contribute to tumor growth. This necessitates dual inhibition of ER-mediated transcription and kinase-mediated growth stimulation for better control of tumor growth (25).

In previous studies involving long-term letrozole-treated tumors and cell lines (LTLT-Ca; ref. 24), ER and aromatase expression were reduced and the mitogen-activated protein kinase signaling pathway was activated. Although fulvestrant did not have a significant effect on the growth of these tumors, ER and growth response to aromatase inhibitors and antiestrogens could be restored in the LTLT-Ca cells by inhibiting the kinase pathway (24). This suggests that combined inhibition of the ER{alpha} and the kinase signaling pathways may provide better control over growth of tumors proliferating in estrogen-deprived conditions.


   Footnotes
 
Grant support: National Cancer Institute, NIH grant CA-62483 (A. Brodie).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Presented in part at the 28th Annual San Antonio Breast Cancer Symposium, December 2005, San Antonio, Texas.

3 Tilghman S, Gauri S, and Brodie A: unpublished data. Back

Received 10/11/06; revised 12/11/06; accepted 1/ 5/07.


   References
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 Abstract
 Materials and Methods
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 References
 

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