Medroxyprogesterone Acetate Induces Cell Proliferation through Up-Regulation of Cyclin D1 Expression via Phosphatidylinositol 3-Kinase/Akt/N
http://www.100md.com
《内分泌学杂志》
Department of Obstetrics and Gynecology, Yamagata University, School of Medicine, Yamagata 990-9585, Japan
Abstract
The mechanism of medroxyprogesterone acetate (MPA)-induced cell proliferation in human breast cancer cells remains elusive. We examined the mechanism by which MPA affects the cyclin D1 expression in progesterone receptor (PR)-positive T47D human breast cancer cells. MPA (10 nM) treatment for 48 h induced proliferation of the cells (1.6-fold induction). MPA induced cyclin D1 expression (3.3-fold induction), and RU486, a selective PR antagonist, blocked the MPA-induced cell proliferation and cyclin D1 expression (23% inhibition). MPA increased both the protein level (2.2-fold induction) and promoter activity (2.7-fold induction) of cyclin D1 in MCF-7 cells transfected with PRB but not with PRA. Although MPA transcriptionally activated cyclin D1 expression, cyclin D1 promoter does not have progesterone-responsive element-related sequence. We further examined the mechanism for the regulation of the cyclin D1 expression. Because the cyclin D1 promoter contains three putative nuclear factor-B (NFB)-binding motifs and NFB is a substrate of Akt, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)/Akt/NFB cascade on the responses of cyclin D1 to MPA. MPA induced the transient phosphorylation of Akt (2.7-fold induction at 5 min), and treatment with PI3K inhibitor (wortmannin) attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) and cell proliferation (40% inhibition). MPA also induced phosphorylation of inhibitor of NFB (IB) (2.3-fold induction), and treatment with wortmannin attenuated the MPA-induced IB phosphorylation (60% inhibition). Treatment with an IB phosphorylation inhibitor (BAY 11-7085) or a specific NFB nuclear translocation inhibitor (SN-50) attenuated the MPA-induced up-regulation of both cyclin D1 expression (80 and 50% inhibition, respectively) and cell proliferation (55 and 34% inhibition, respectively). Because MPA induced a transient phosphorylation of Akt and the cyclin D1 promoter contains no progesterone-responsive element-related sequence, the MPA-induced cell proliferation through PRB by up-regulation of cyclin D1 expression via the PI3K/Akt/NFB cascade may be a nongenomic mechanism.
Introduction
THE FEMALE SEX steroid progesterone and its synthetic analogs, progestins, have complex effects on cell proliferation that depend on the cell cycle, tissue, and treatment regimen (1). For example, in the uterus progesterone acts synergistically with estrogen to stimulate stromal proliferation but inhibits estrogen-induced epithelial proliferation. The latter effect has led to the addition of progestin to estrogen replacement therapies to counteract the increased risk of endometrial cancer arising from treatment with estrogen alone (2). Although synthetic progestins have an established role in the therapy of endometrial cancer (2, 3), whether progesterone has a stimulatory or inhibitory effect on the proliferation of normal breast epithelium remains controversial. In the Women’s Health Initiative’s (WHI) large prospective, randomized, controlled study, although women on the conjugated equine estrogens (CEE)-medroxyprogesterone acetate (MPA) arm had an increase in the relative risks for cardiovascular events and breast cancer (4), the more recent reports on women on the CEE-only treatment arm did not show an adverse effect of estrogen on the breast cancer (5). Thus, there is a possibility that progestin has a carcinogenetic effect on breast epithelium.
Progestins have a biphasic effect on the proliferation of breast cancer cells in culture (6). Studies focusing on the stimulatory component of the response showed that the entry of progestin-stimulated cells into S phase was preceded by transient increases in c-myc and cyclin D1 mRNA abundance (6). After 6 h of progestin treatment, cyclin D1 induction reached a maximum of 3- to 4-fold but declined thereafter. The induction of cyclin D1 led to increases in the abundance of cyclin D1-cyclin-dependent kinase 4 complex, cyclin D1-cyclin-dependent kinase 4 activity, and the relative amount of the hyperphosphorylated form of retinoblastoma proteins (7).
The cyclin D1 promoter contains multiple regulatory elements [transcriptional regulatory element, E2F, octamer transcription factor, specificity protein 1, cAMP response element, and nuclear factor-B (NFB)] (8) and some potential elements that may be involved in the transcriptional regulation of the gene. However, no progesterone-responsive element (PRE)-related sequence has been identified in or proximal to the cyclin D1 promoter. It was reported that the cyclin D1 promoter contains three putative NFB-binding motifs (9, 10, 11). In addition, the existence of a nongenomic signaling pathway of progestin has been reported (12). This pathway dose not involve the classical nuclear transcription factor progesterone receptor (PR). The phosphatidylinositol 3-kinase (PI3K)/Akt/NFB cascade has been reported to be involved in the estrogen-induced cyclin D1 expression in human breast cancer cells (13). We recently reported that the PI3K/Akt/NFB cascade is involved in the estrogen-induced telomerase activity in human breast cancer cells (14) and human ovarian cancer cells (15). In addition, we have reported that the PI3K/Akt cascade is involved in the estrogen-induced endothelial nitric oxide synthase activity in vascular endothelial cells (16). Thus, the PI3K/Akt/NFB cascade appears to have an important role in several nongenomic actions of estrogen. It is possible that progestin also enhances the transcription of cyclin D1 via the PI3/Akt/NFB cascade.
There are two isoforms of PRs, PRA and PRB. The PRA and PRB isoforms of PR exist naturally and are transcribed by the two promoters of a single gene (17). PRA and PRB have different physiological functions, and their ratio varies widely in breast cancers. Because PRA and PRB have different molecular functions, it may be important to evaluate which isoform of PR is involved in the progestin-induced proliferation of breast cancer cells.
These considerations led us to examine the mechanism by which progestin induces cell proliferation and cyclin D1 expression via a PI3K/Akt/NFB cascade in T47D cells, a human breast cancer cell line that expresses progesterone receptors. In addition, we examined which PR is critical in MPA-induced cyclin D1 expression and whether this MPA effect is a nongenomic action.
Materials and Methods
Materials
17-Estradiol and medroxyprogesterone acetate were purchased from Sigma (St. Louis, MO). The anti-cyclin D1 antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), the -tubulin antibody was obtained from Calbiochem (La Jolla, CA), and the anti-phospho-Akt, Akt, and phospho-inhibitory NFB (IB) antibodies were obtained from Cell Signaling (Beverly, MA). The PR antagonist RU486 was purchased from Sigma. The IB phosphorylation inhibitor BAY-11–7082 was purchased from Alexis Biochemicals (San Diego, CA). The specific NFB nuclear translocation inhibitor SN-50 was purchased from BIOMOL (Plymouth Meeting, PA).
Cell cultures
T47D cells obtained from Dr. Satoru Kyo (Kanazawa University Medical School) were incubated in DMEM containing 10% fetal calf serum (FCS) and 10 μg/ml insulin, L-glutamine, penicillin streptomycin under a 5% CO2 atmosphere at 37 C. MCF7 cells obtained from the American Type Culture Collection (Manassas, VA) were incubated in DMEM containing 10% FCS and L-glutamine, penicillin streptomycin under a 5% CO2 atmosphere at 37 C. To assay estrogen or MPA induction, the cells were cultured in medium containing 10% dextran-coated charcoal-treated FCS for 24–48 h before hormone exposure.
Cell proliferation assay
The cells were plated at a density of 10 or 40 x 104 cells/well in 12-well plates and allowed to attach overnight. The cells were then growth arrested by incubation in phenol red-free DMEM with 10% charcoal-stripped serum for 24 h followed by treatment with vehicle or MPA by exchanging the culture medium containing these agents with fresh medium every 24 h for 4 d. A Neubauer chamber was used to count cells, and trypan blue experiments were carried out in quadruplicate.
Western blot analysis
The cells were incubated in phenol red-free medium without serum for 24 h and subsequently treated with various agents. They were then washed twice with phosphate-buffered saline and lysed in ice-cold buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 μM sodium orthovanadate, 100 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride]. The lysates were centrifuged at 12,000 x g at 4 C for 15 min, and the protein concentrations of the supernatants were measured using a protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blocking was done in 10% BSA in 1 x Tris-buffered saline. Western blot analysis was performed with various specific primary antibodies.
Transfection of MCF-7 cells with human PR cDNAs
The MCF-7 cells were grown routinely in 2-cm dishes in DMEM containing 10% FCS. One microgram pSG5-hPR, form A and B PR obtained by Dr. Fernando Auricchio (II Universita di Napoli, Napoli, Italy) (18), was transiently transfected into MCF-7 cells using LipofectAMINE Plus (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. As a control, parallel MCF-7 aliquots were also transfected with 1 μg of pSG5 vector alone.
Luciferase assay
2x PRE-thymidine kinase (TK)-luciferase (LUC), containing two copies of a consensus PRE upstream of the thymidine kinase promoter, obtained from Dr. Donald P. McDonnell (Duke University, Durham, NC) (19) or the reporter construct cyclin-LUC containing a 1879-bp fragment encompassing positions –1745 to +134 of the human cyclin D1 gene ligated upstream of the luciferase reporter gene in pA3 obtained from Pestell and colleagues (11) was transiently transfected into T47D cells or MCF-7 cells transfected with PRA or PRB for 24 h using LipofectAMINE Plus (Life Technologies, Gaithersburg, MD) according to the manufacturer’s protocol. Cells were harvested and subjected to luciferase assays using the luciferase assay system (Promega, San Luis Obispo, CA) as described previously (20). A plasmid expressing the bacterial -galactosidase gene was also cotransfected in each experiment to serve as an internal control for transfection efficiency.
Statistics
Statistical analysis was performed using one-way ANOVA followed by Bonferroni test, and P < 0.05 was considered significant. Data are expressed as the mean ± SE.
Results
Effects of progestin (MPA) on the proliferation of T47D breast cancer cells
We first examined whether MPA regulates the proliferation of PR-positive T47D human breast cancer cells (Fig. 1A). MPA (10 nM) treatment for 48 h resulted in an increase in the number of cells (1.6-fold induction). However, the MPA effect was followed by an inhibition of proliferation after 72 h (Fig. 1A) as previously reported (21). The optimal concentration of MPA was 10 nM; no significant effect was induced by 1 nM MPA, and no more proliferative effect was obtained by 100 nM MPA (data not shown).
Cyclin D1 expression after MPA stimulation
We next examined the effects of MPA on cyclin D1 expression. T47D cells were incubated with or without 10 nM MPA for the indicated times (Fig. 1B) or with the indicated concentrations of MPA for 4 h (Fig. 1C) and then used to prepare lysates that were subjected to Western blotting with anti-cyclin D1 or anti--tubulin antibody (Fig. 1, B and C). Although MPA did not affect the expression of -tubulin, it increased the expression of cyclin D1 at 2 h (2.8-fold induction), followed by a plateau at 4 h (3.3-fold induction) and a decline thereafter (Fig. 1B). The maximum increase of the expression of cyclin D1 by MPA was detected at a dose of 10 nM (Fig. 1C), which corresponds to the physiological progesterone levels (21).
To examine whether the MPA-induced cell proliferation and up-regulation of cyclin D1 expression are mediated via PR, T47D cells were treated with RU486, a selective PR antagonist. Cotreatment of cells with 1 μM RU486 together with MPA blocked the MPA-induced cell proliferation (23% inhibition) (Fig. 2A). The MPA-induced cyclin D1 expression was also significantly inhibited by cotreatment with RU486 in a dose-dependent manner (Fig. 2B).
Next, the role of PRA or PRB in the MPA-induced cyclin D1 expression was evaluated. BecauseT47D cells express both PRA and PRB, PR-negative MCF-7 human breast cancer cells transfected with human (h) PRA, hPRB, or the vehicle (pSG5) were used. The expression of PRA and PRB was confirmed by Western blotting with anti-PR antibodies in MCF-7 cells transfected with hPRA or hPRB (Fig. 3A). Transcriptional activation of PRE by MPA was examined using MCF-7 cells transiently transfected with 2 x PRE-TK-LUC reporter plasmid and with hPRA, hPRB, or vehicle (Fig. 3B). MPA significantly activated the 2 x PRE-TK-LUC gene in MCF-7 cells transfected with hPRA, hPRB, and also in T47D cells, which express both types of PR. Estrogen receptor- is expressed in MCF-7 cells (22), and estrogen significantly induced the expression of cyclin D1 in MCF-7 cells transfected with vehicle (pSG5), hPRA, or hPRB (Fig. 3C). Interestingly, MPA induced the expression of cyclin D1 in MCF-7 cells transfected with PRB (2.2-fold induction) but not in cells transfected with PRA (Fig. 3C). To examine whether the MPA-induced cyclin D1 protein expression is due to transcriptional regulation of the cyclin D1 promoter, the regulation of the cyclin D1 promoter in MCF-7 cells transfected with PRA or PRB was investigated using transient transfection with a luciferase plasmid containing the human cyclin D1 promoter. Similar to the differential effects described above, MPA induced an increase of cyclin D1 promoter activity in MCF-7 cells transfected with PRB (2.7-fold induction) but not in cells transfected with PRA (Fig. 3D). MPA also increased the cyclin D1 promoter activity in T47D cells. These results indicate that MPA induces the cyclin D1 expression through PRB.
MPA-induced Akt phosphorylation
Because it was previously reported that cyclin D1 expression is induced via an Akt cascade (13), we examined whether MPA induces Akt phosphorylation in T47D cells. The cells were treated with 10 nM MPA for various times and then used to prepare lysates that were subjected to Western blotting with anti-phospho-Akt or -Akt antibody. Although MPA did not affect the expression of Akt (Fig. 4A, bottom panel), it transiently induced phosphorylation of Akt (2.7-fold induction at 5 min) (Fig. 4A, upper and middle panels). To examine whether the MPA-induced Akt phosphorylation is mediated via PR, T47D cells were treated with RU486. The MPA-induced Akt phosphorylation was inhibited by treatment with 1 μM RU486 (42% inhibition) (Fig. 4B), indicating that MPA-induced Akt phosphorylation is mediated via PR. We then examined whether MPA induces cyclin D1 expression via an Akt cascade. The effects of PI3K inhibitor (wortmannin) on the MPA-induced up-regulation of cyclin D1 expression were examined. Although wortmannin had no effect on the expression of -tubulin (Fig. 4C, bottom panel), it attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) (Fig. 4C, upper and middle panels). Moreover, we examined whether MPA induces cell proliferation via an Akt cascade. Although wortmannin alone did not exhibit a significant effect, it attenuated the MPA-induced cell proliferation (40% inhibition) (Fig. 4D). These results suggest that an Akt cascade is involved in the MPA-induced up-regulation of cyclin D1 expression and cell proliferation.
MPA-induced cyclin D1 expression via PI3K/Akt/NFB cascade
Via the phosphorylation of IB kinase, Akt also activates NFB, a transcription factor that has been implicated in cell survival (23, 24). In addition, it has been previously reported that the cyclin D1 promoter contains three putative NFB-binding motifs (9, 10, 11). Therefore, we examined whether NFB is a nuclear target in the MPA-induced up-regulation of cyclin D1 expression via an Akt cascade. NFB is regulated through its association with an inhibitory cofactor, IB, which sequesters NFB in the cytoplasm. Phosphorylation of IB by upstream kinases promotes its degradation, allowing NFB to translocate to the nucleus and induce target genes (23, 24). We examined whether MPA induces the phosphorylation of IB (Fig. 5A). Cells were treated with MPA for the indicated times and used to prepare lysates that were analyzed by Western blotting with anti-phospho-IB, or anti-Akt antibody. Although the expression of Akt was not changed (Fig. 5A, bottom panel), MPA-stimulated T47D cells showed a transient increase in the phosphorylation of IB (2.3-fold induction) (Fig. 5A, upper and middle panels). Cotreatment with 1 μM RU486 together with MPA significantly attenuated the MPA-induced phosphorylation of IB (21% inhibition) (Fig. 5B, upper and middle panels). Moreover, pretreatment with wortmannin significantly attenuated the MPA-induced phosphorylation of IB (60% inhibition) (Fig. 5C, upper and middle panels). These results suggest that MPA induces the NFB cascade through PR via the PI3K/Akt cascade. The effects of IB phosphorylation inhibitor (BAY 11–7085) (25) or a specific NFB nuclear translocation inhibitor (SN-50) (26) on both MPA-induced up-regulation of cyclin D1 expression and cell proliferation were examined. Although SN-50 (Fig. 6C, bottom panel) did not have an effect on the expression of -tubulin, both BAY 11–7085 (Fig. 6A, upper and middle panels) and SN-50 (Fig. 6C, upper and middle panels) significantly attenuated the MPA-induced up-regulation of cyclin D1 expression (80 and 50% inhibition, respectively). Moreover, both BAY 11–7085 (Fig. 6B) and SN-50 (Fig. 6D) significantly attenuated the MPA-induced cell proliferation (55 and 34% inhibition, respectively). These results suggest that MPA induces cell proliferation through up-regulation of cyclin D1 expression via the PI3K/Akt/NFB cascade.
Discussion
Histological studies revealed that the prognosis of breast cancer depends on the expression of PR as well as on the expression of estrogen receptor (27). In addition, clinical observational studies also revealed that the addition of progestin to estrogen increased the risk of breast cancer (28, 29, 30, 31, 32, 33, 34). In the WHI, a large prospective, randomized, controlled study, although women on estrogen alone did not show an increase in the risk for breast cancer (5), women on combined estrogen-progestin showed an increase in the risk for breast cancer (4). Thus, there is a possibility that progestin has a carcinogenetic effect on the breast epithelium.
Progestins are known to have a biphasic effect on the proliferation of breast cancer cells in culture (6). It was reported that an increase in the proliferative activity of T47D cells after 24 and 48 h of MPA treatment was followed by an inhibition of proliferation after 72 h (21). In addition, the expression of cyclin D1 showed a similar biphasic response in this context (21), suggesting that cyclin D1 may be a mediator of these effects. We also detected a biphasic effect of MPA on cell proliferation (Fig. 1A) and cyclin D1 expression (Fig. 1B) because we previously detected a biphasic effect of MPA on human telomerase reverse transcriptase gene expression (35). Although future studies will be necessary to dissect the specific molecular mechanisms responsible for this biphasic effect of MPA, cyclin D1 expression might be considered at least to be an important mediator of the biphasic proliferative effect of MPA.
The expression of cyclin D1 is reported to be involved in the induction of cell proliferation by MPA in human breast cancer cells (14, 36). However, the mechanism of MPA-induced cyclin D1 expression remains unclear. Novel mechanisms of signal transduction have been discovered for PRs in various tissues, some of which are independent of gene transcription regulation and are therefore termed nongenomic (37, 38, 39). MPA-induced Akt phosphorylation within 5 min (Fig. 4) and IB phosphorylation within 60 min (Fig. 5), indicating a rapid response to MPA. In addition, no PRE-related sequence has been identified in the proximal cyclin D1 promoter. It has been reported that the cyclin D1 promoter contains three putative NFB-binding motifs (9, 10, 11). These facts suggest that the MPA-induced cyclin D1 expression is regulated by the PI3K/Akt/NFB cascade in a nongenomic manner.
There are two isoforms of PR, PRA and PRB. What are the different roles of PRA and PRB in the biological actions of progestin It has been reported that an imbalance in the native ratio of the two isoforms can lead to alterations in PR signaling (40) and mammary gland development (41). For example, PRB is required for progestin-dependent induction of vascular endothelial growth factor (42). Recently the presence of a specific membrane-bound PR in mature human spermatozoa was shown to regulate important sperm functions (43). PRB is also involved in the MPA-induced cyclin D1 expression via the ERK cascade (18) as well as involved in the MPA-induced cyclin D1 expression via the PI3K/Akt/NFB cascade, as detected in this study. Because blocking the ERK cascade with PD98059 had no effect on the MPA-induced Akt phosphorylation (data not shown), we believe that there is no cross-talk between the PI3K/Akt/NFB cascade and ERK cascade. Thus, because PRB induces both the PI3K/Akt/NFB and ERK cascade, there is a possibility that PRB localized in the membrane regulates cell proliferation in the mammary gland. However, the function of PRA remains unclear, and additional detailed examinations will be required to clarify it.
A network of interactions among PR signaling, type I IGF receptor (IGF-IR) signaling, and heregulin/ErbB-2 signaling was reported (45). Although MPA induces the phosphorylation of IGF-IR and ErbB-2, and IGF-IR directs ErbB-2 phosphorylation, blockage of either IGF-IR or ErbB-2 had no effect on the MPA-induced proliferation of breast cancer cells (44). Moreover, inhibition of breast cancer proliferation by blockage of IGF-IR involves inactivation of ErbB-2, PI3K/Akt, and MAPK signaling pathways but not modulation of PR activity, indicating that PR function remains unaffected by targeted blockage of IGF-IR (45). Thus, because cyclin D1 induction via the PI3K/Akt/NFB cascade seems to be necessary for the MPA-induced proliferation of breast cancer cells, it is important to investigate whether IGF-IR and/or ErbBs are involved in cyclin D1 induction via the PI3K/Akt/NFB cascade. We are currently examining this issue.
In the WHI study, although women on the CEE-only treatment arm did not show an increase in the risk for breast cancer (5), women on the CEE-MPA arm showed an increase in the risk for breast cancer (4). Are the mechanisms of estrogen- and progestin-induced cyclin D1 expression different The expression of cyclin D1 did not synergistically increase in the cells treated with both MPA and estrogen (data not shown), suggesting that the signaling cascade of MPA-induced cyclin D1 expression might be similar to that of estrogen-induced cyclin D1 expression. However, because it is known that the expression of PR is enhanced by the transcriptional activation by estrogen in the uterine endometrium (46) and mammary gland (47), estrogen priming may enhance the signaling cascade of PR. Further investigations will be necessary to explain the findings of the WHI study. Because the effect of MPA on the cell proliferation was examined here in human breast cancer cells, it is not yet possible to say whether progestin has a carcinogenetic effect on the normal breast epithelium. We are currently examining the effect of MPA on carcinogenesis using primary cultures of normal breast epithelium.
Footnotes
Abbreviations: CEE, Conjugated equine estrogens; FCS, fetal calf serum; h, human; IB, inhibitor of NFB; IGF-IR, type I IGF receptor; LUC, luciferase; MPA, medroxyprogesterone acetate; NFB, nuclear factor B; PI3K, phosphatidylinositol 3-kinase; PR, progesterone receptor; PRE, progesterone-responsive element; SDS, sodium dodecyl sulfate; TK, thymidine kinase.
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Abstract
The mechanism of medroxyprogesterone acetate (MPA)-induced cell proliferation in human breast cancer cells remains elusive. We examined the mechanism by which MPA affects the cyclin D1 expression in progesterone receptor (PR)-positive T47D human breast cancer cells. MPA (10 nM) treatment for 48 h induced proliferation of the cells (1.6-fold induction). MPA induced cyclin D1 expression (3.3-fold induction), and RU486, a selective PR antagonist, blocked the MPA-induced cell proliferation and cyclin D1 expression (23% inhibition). MPA increased both the protein level (2.2-fold induction) and promoter activity (2.7-fold induction) of cyclin D1 in MCF-7 cells transfected with PRB but not with PRA. Although MPA transcriptionally activated cyclin D1 expression, cyclin D1 promoter does not have progesterone-responsive element-related sequence. We further examined the mechanism for the regulation of the cyclin D1 expression. Because the cyclin D1 promoter contains three putative nuclear factor-B (NFB)-binding motifs and NFB is a substrate of Akt, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)/Akt/NFB cascade on the responses of cyclin D1 to MPA. MPA induced the transient phosphorylation of Akt (2.7-fold induction at 5 min), and treatment with PI3K inhibitor (wortmannin) attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) and cell proliferation (40% inhibition). MPA also induced phosphorylation of inhibitor of NFB (IB) (2.3-fold induction), and treatment with wortmannin attenuated the MPA-induced IB phosphorylation (60% inhibition). Treatment with an IB phosphorylation inhibitor (BAY 11-7085) or a specific NFB nuclear translocation inhibitor (SN-50) attenuated the MPA-induced up-regulation of both cyclin D1 expression (80 and 50% inhibition, respectively) and cell proliferation (55 and 34% inhibition, respectively). Because MPA induced a transient phosphorylation of Akt and the cyclin D1 promoter contains no progesterone-responsive element-related sequence, the MPA-induced cell proliferation through PRB by up-regulation of cyclin D1 expression via the PI3K/Akt/NFB cascade may be a nongenomic mechanism.
Introduction
THE FEMALE SEX steroid progesterone and its synthetic analogs, progestins, have complex effects on cell proliferation that depend on the cell cycle, tissue, and treatment regimen (1). For example, in the uterus progesterone acts synergistically with estrogen to stimulate stromal proliferation but inhibits estrogen-induced epithelial proliferation. The latter effect has led to the addition of progestin to estrogen replacement therapies to counteract the increased risk of endometrial cancer arising from treatment with estrogen alone (2). Although synthetic progestins have an established role in the therapy of endometrial cancer (2, 3), whether progesterone has a stimulatory or inhibitory effect on the proliferation of normal breast epithelium remains controversial. In the Women’s Health Initiative’s (WHI) large prospective, randomized, controlled study, although women on the conjugated equine estrogens (CEE)-medroxyprogesterone acetate (MPA) arm had an increase in the relative risks for cardiovascular events and breast cancer (4), the more recent reports on women on the CEE-only treatment arm did not show an adverse effect of estrogen on the breast cancer (5). Thus, there is a possibility that progestin has a carcinogenetic effect on breast epithelium.
Progestins have a biphasic effect on the proliferation of breast cancer cells in culture (6). Studies focusing on the stimulatory component of the response showed that the entry of progestin-stimulated cells into S phase was preceded by transient increases in c-myc and cyclin D1 mRNA abundance (6). After 6 h of progestin treatment, cyclin D1 induction reached a maximum of 3- to 4-fold but declined thereafter. The induction of cyclin D1 led to increases in the abundance of cyclin D1-cyclin-dependent kinase 4 complex, cyclin D1-cyclin-dependent kinase 4 activity, and the relative amount of the hyperphosphorylated form of retinoblastoma proteins (7).
The cyclin D1 promoter contains multiple regulatory elements [transcriptional regulatory element, E2F, octamer transcription factor, specificity protein 1, cAMP response element, and nuclear factor-B (NFB)] (8) and some potential elements that may be involved in the transcriptional regulation of the gene. However, no progesterone-responsive element (PRE)-related sequence has been identified in or proximal to the cyclin D1 promoter. It was reported that the cyclin D1 promoter contains three putative NFB-binding motifs (9, 10, 11). In addition, the existence of a nongenomic signaling pathway of progestin has been reported (12). This pathway dose not involve the classical nuclear transcription factor progesterone receptor (PR). The phosphatidylinositol 3-kinase (PI3K)/Akt/NFB cascade has been reported to be involved in the estrogen-induced cyclin D1 expression in human breast cancer cells (13). We recently reported that the PI3K/Akt/NFB cascade is involved in the estrogen-induced telomerase activity in human breast cancer cells (14) and human ovarian cancer cells (15). In addition, we have reported that the PI3K/Akt cascade is involved in the estrogen-induced endothelial nitric oxide synthase activity in vascular endothelial cells (16). Thus, the PI3K/Akt/NFB cascade appears to have an important role in several nongenomic actions of estrogen. It is possible that progestin also enhances the transcription of cyclin D1 via the PI3/Akt/NFB cascade.
There are two isoforms of PRs, PRA and PRB. The PRA and PRB isoforms of PR exist naturally and are transcribed by the two promoters of a single gene (17). PRA and PRB have different physiological functions, and their ratio varies widely in breast cancers. Because PRA and PRB have different molecular functions, it may be important to evaluate which isoform of PR is involved in the progestin-induced proliferation of breast cancer cells.
These considerations led us to examine the mechanism by which progestin induces cell proliferation and cyclin D1 expression via a PI3K/Akt/NFB cascade in T47D cells, a human breast cancer cell line that expresses progesterone receptors. In addition, we examined which PR is critical in MPA-induced cyclin D1 expression and whether this MPA effect is a nongenomic action.
Materials and Methods
Materials
17-Estradiol and medroxyprogesterone acetate were purchased from Sigma (St. Louis, MO). The anti-cyclin D1 antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), the -tubulin antibody was obtained from Calbiochem (La Jolla, CA), and the anti-phospho-Akt, Akt, and phospho-inhibitory NFB (IB) antibodies were obtained from Cell Signaling (Beverly, MA). The PR antagonist RU486 was purchased from Sigma. The IB phosphorylation inhibitor BAY-11–7082 was purchased from Alexis Biochemicals (San Diego, CA). The specific NFB nuclear translocation inhibitor SN-50 was purchased from BIOMOL (Plymouth Meeting, PA).
Cell cultures
T47D cells obtained from Dr. Satoru Kyo (Kanazawa University Medical School) were incubated in DMEM containing 10% fetal calf serum (FCS) and 10 μg/ml insulin, L-glutamine, penicillin streptomycin under a 5% CO2 atmosphere at 37 C. MCF7 cells obtained from the American Type Culture Collection (Manassas, VA) were incubated in DMEM containing 10% FCS and L-glutamine, penicillin streptomycin under a 5% CO2 atmosphere at 37 C. To assay estrogen or MPA induction, the cells were cultured in medium containing 10% dextran-coated charcoal-treated FCS for 24–48 h before hormone exposure.
Cell proliferation assay
The cells were plated at a density of 10 or 40 x 104 cells/well in 12-well plates and allowed to attach overnight. The cells were then growth arrested by incubation in phenol red-free DMEM with 10% charcoal-stripped serum for 24 h followed by treatment with vehicle or MPA by exchanging the culture medium containing these agents with fresh medium every 24 h for 4 d. A Neubauer chamber was used to count cells, and trypan blue experiments were carried out in quadruplicate.
Western blot analysis
The cells were incubated in phenol red-free medium without serum for 24 h and subsequently treated with various agents. They were then washed twice with phosphate-buffered saline and lysed in ice-cold buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 μM sodium orthovanadate, 100 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride]. The lysates were centrifuged at 12,000 x g at 4 C for 15 min, and the protein concentrations of the supernatants were measured using a protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blocking was done in 10% BSA in 1 x Tris-buffered saline. Western blot analysis was performed with various specific primary antibodies.
Transfection of MCF-7 cells with human PR cDNAs
The MCF-7 cells were grown routinely in 2-cm dishes in DMEM containing 10% FCS. One microgram pSG5-hPR, form A and B PR obtained by Dr. Fernando Auricchio (II Universita di Napoli, Napoli, Italy) (18), was transiently transfected into MCF-7 cells using LipofectAMINE Plus (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. As a control, parallel MCF-7 aliquots were also transfected with 1 μg of pSG5 vector alone.
Luciferase assay
2x PRE-thymidine kinase (TK)-luciferase (LUC), containing two copies of a consensus PRE upstream of the thymidine kinase promoter, obtained from Dr. Donald P. McDonnell (Duke University, Durham, NC) (19) or the reporter construct cyclin-LUC containing a 1879-bp fragment encompassing positions –1745 to +134 of the human cyclin D1 gene ligated upstream of the luciferase reporter gene in pA3 obtained from Pestell and colleagues (11) was transiently transfected into T47D cells or MCF-7 cells transfected with PRA or PRB for 24 h using LipofectAMINE Plus (Life Technologies, Gaithersburg, MD) according to the manufacturer’s protocol. Cells were harvested and subjected to luciferase assays using the luciferase assay system (Promega, San Luis Obispo, CA) as described previously (20). A plasmid expressing the bacterial -galactosidase gene was also cotransfected in each experiment to serve as an internal control for transfection efficiency.
Statistics
Statistical analysis was performed using one-way ANOVA followed by Bonferroni test, and P < 0.05 was considered significant. Data are expressed as the mean ± SE.
Results
Effects of progestin (MPA) on the proliferation of T47D breast cancer cells
We first examined whether MPA regulates the proliferation of PR-positive T47D human breast cancer cells (Fig. 1A). MPA (10 nM) treatment for 48 h resulted in an increase in the number of cells (1.6-fold induction). However, the MPA effect was followed by an inhibition of proliferation after 72 h (Fig. 1A) as previously reported (21). The optimal concentration of MPA was 10 nM; no significant effect was induced by 1 nM MPA, and no more proliferative effect was obtained by 100 nM MPA (data not shown).
Cyclin D1 expression after MPA stimulation
We next examined the effects of MPA on cyclin D1 expression. T47D cells were incubated with or without 10 nM MPA for the indicated times (Fig. 1B) or with the indicated concentrations of MPA for 4 h (Fig. 1C) and then used to prepare lysates that were subjected to Western blotting with anti-cyclin D1 or anti--tubulin antibody (Fig. 1, B and C). Although MPA did not affect the expression of -tubulin, it increased the expression of cyclin D1 at 2 h (2.8-fold induction), followed by a plateau at 4 h (3.3-fold induction) and a decline thereafter (Fig. 1B). The maximum increase of the expression of cyclin D1 by MPA was detected at a dose of 10 nM (Fig. 1C), which corresponds to the physiological progesterone levels (21).
To examine whether the MPA-induced cell proliferation and up-regulation of cyclin D1 expression are mediated via PR, T47D cells were treated with RU486, a selective PR antagonist. Cotreatment of cells with 1 μM RU486 together with MPA blocked the MPA-induced cell proliferation (23% inhibition) (Fig. 2A). The MPA-induced cyclin D1 expression was also significantly inhibited by cotreatment with RU486 in a dose-dependent manner (Fig. 2B).
Next, the role of PRA or PRB in the MPA-induced cyclin D1 expression was evaluated. BecauseT47D cells express both PRA and PRB, PR-negative MCF-7 human breast cancer cells transfected with human (h) PRA, hPRB, or the vehicle (pSG5) were used. The expression of PRA and PRB was confirmed by Western blotting with anti-PR antibodies in MCF-7 cells transfected with hPRA or hPRB (Fig. 3A). Transcriptional activation of PRE by MPA was examined using MCF-7 cells transiently transfected with 2 x PRE-TK-LUC reporter plasmid and with hPRA, hPRB, or vehicle (Fig. 3B). MPA significantly activated the 2 x PRE-TK-LUC gene in MCF-7 cells transfected with hPRA, hPRB, and also in T47D cells, which express both types of PR. Estrogen receptor- is expressed in MCF-7 cells (22), and estrogen significantly induced the expression of cyclin D1 in MCF-7 cells transfected with vehicle (pSG5), hPRA, or hPRB (Fig. 3C). Interestingly, MPA induced the expression of cyclin D1 in MCF-7 cells transfected with PRB (2.2-fold induction) but not in cells transfected with PRA (Fig. 3C). To examine whether the MPA-induced cyclin D1 protein expression is due to transcriptional regulation of the cyclin D1 promoter, the regulation of the cyclin D1 promoter in MCF-7 cells transfected with PRA or PRB was investigated using transient transfection with a luciferase plasmid containing the human cyclin D1 promoter. Similar to the differential effects described above, MPA induced an increase of cyclin D1 promoter activity in MCF-7 cells transfected with PRB (2.7-fold induction) but not in cells transfected with PRA (Fig. 3D). MPA also increased the cyclin D1 promoter activity in T47D cells. These results indicate that MPA induces the cyclin D1 expression through PRB.
MPA-induced Akt phosphorylation
Because it was previously reported that cyclin D1 expression is induced via an Akt cascade (13), we examined whether MPA induces Akt phosphorylation in T47D cells. The cells were treated with 10 nM MPA for various times and then used to prepare lysates that were subjected to Western blotting with anti-phospho-Akt or -Akt antibody. Although MPA did not affect the expression of Akt (Fig. 4A, bottom panel), it transiently induced phosphorylation of Akt (2.7-fold induction at 5 min) (Fig. 4A, upper and middle panels). To examine whether the MPA-induced Akt phosphorylation is mediated via PR, T47D cells were treated with RU486. The MPA-induced Akt phosphorylation was inhibited by treatment with 1 μM RU486 (42% inhibition) (Fig. 4B), indicating that MPA-induced Akt phosphorylation is mediated via PR. We then examined whether MPA induces cyclin D1 expression via an Akt cascade. The effects of PI3K inhibitor (wortmannin) on the MPA-induced up-regulation of cyclin D1 expression were examined. Although wortmannin had no effect on the expression of -tubulin (Fig. 4C, bottom panel), it attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) (Fig. 4C, upper and middle panels). Moreover, we examined whether MPA induces cell proliferation via an Akt cascade. Although wortmannin alone did not exhibit a significant effect, it attenuated the MPA-induced cell proliferation (40% inhibition) (Fig. 4D). These results suggest that an Akt cascade is involved in the MPA-induced up-regulation of cyclin D1 expression and cell proliferation.
MPA-induced cyclin D1 expression via PI3K/Akt/NFB cascade
Via the phosphorylation of IB kinase, Akt also activates NFB, a transcription factor that has been implicated in cell survival (23, 24). In addition, it has been previously reported that the cyclin D1 promoter contains three putative NFB-binding motifs (9, 10, 11). Therefore, we examined whether NFB is a nuclear target in the MPA-induced up-regulation of cyclin D1 expression via an Akt cascade. NFB is regulated through its association with an inhibitory cofactor, IB, which sequesters NFB in the cytoplasm. Phosphorylation of IB by upstream kinases promotes its degradation, allowing NFB to translocate to the nucleus and induce target genes (23, 24). We examined whether MPA induces the phosphorylation of IB (Fig. 5A). Cells were treated with MPA for the indicated times and used to prepare lysates that were analyzed by Western blotting with anti-phospho-IB, or anti-Akt antibody. Although the expression of Akt was not changed (Fig. 5A, bottom panel), MPA-stimulated T47D cells showed a transient increase in the phosphorylation of IB (2.3-fold induction) (Fig. 5A, upper and middle panels). Cotreatment with 1 μM RU486 together with MPA significantly attenuated the MPA-induced phosphorylation of IB (21% inhibition) (Fig. 5B, upper and middle panels). Moreover, pretreatment with wortmannin significantly attenuated the MPA-induced phosphorylation of IB (60% inhibition) (Fig. 5C, upper and middle panels). These results suggest that MPA induces the NFB cascade through PR via the PI3K/Akt cascade. The effects of IB phosphorylation inhibitor (BAY 11–7085) (25) or a specific NFB nuclear translocation inhibitor (SN-50) (26) on both MPA-induced up-regulation of cyclin D1 expression and cell proliferation were examined. Although SN-50 (Fig. 6C, bottom panel) did not have an effect on the expression of -tubulin, both BAY 11–7085 (Fig. 6A, upper and middle panels) and SN-50 (Fig. 6C, upper and middle panels) significantly attenuated the MPA-induced up-regulation of cyclin D1 expression (80 and 50% inhibition, respectively). Moreover, both BAY 11–7085 (Fig. 6B) and SN-50 (Fig. 6D) significantly attenuated the MPA-induced cell proliferation (55 and 34% inhibition, respectively). These results suggest that MPA induces cell proliferation through up-regulation of cyclin D1 expression via the PI3K/Akt/NFB cascade.
Discussion
Histological studies revealed that the prognosis of breast cancer depends on the expression of PR as well as on the expression of estrogen receptor (27). In addition, clinical observational studies also revealed that the addition of progestin to estrogen increased the risk of breast cancer (28, 29, 30, 31, 32, 33, 34). In the WHI, a large prospective, randomized, controlled study, although women on estrogen alone did not show an increase in the risk for breast cancer (5), women on combined estrogen-progestin showed an increase in the risk for breast cancer (4). Thus, there is a possibility that progestin has a carcinogenetic effect on the breast epithelium.
Progestins are known to have a biphasic effect on the proliferation of breast cancer cells in culture (6). It was reported that an increase in the proliferative activity of T47D cells after 24 and 48 h of MPA treatment was followed by an inhibition of proliferation after 72 h (21). In addition, the expression of cyclin D1 showed a similar biphasic response in this context (21), suggesting that cyclin D1 may be a mediator of these effects. We also detected a biphasic effect of MPA on cell proliferation (Fig. 1A) and cyclin D1 expression (Fig. 1B) because we previously detected a biphasic effect of MPA on human telomerase reverse transcriptase gene expression (35). Although future studies will be necessary to dissect the specific molecular mechanisms responsible for this biphasic effect of MPA, cyclin D1 expression might be considered at least to be an important mediator of the biphasic proliferative effect of MPA.
The expression of cyclin D1 is reported to be involved in the induction of cell proliferation by MPA in human breast cancer cells (14, 36). However, the mechanism of MPA-induced cyclin D1 expression remains unclear. Novel mechanisms of signal transduction have been discovered for PRs in various tissues, some of which are independent of gene transcription regulation and are therefore termed nongenomic (37, 38, 39). MPA-induced Akt phosphorylation within 5 min (Fig. 4) and IB phosphorylation within 60 min (Fig. 5), indicating a rapid response to MPA. In addition, no PRE-related sequence has been identified in the proximal cyclin D1 promoter. It has been reported that the cyclin D1 promoter contains three putative NFB-binding motifs (9, 10, 11). These facts suggest that the MPA-induced cyclin D1 expression is regulated by the PI3K/Akt/NFB cascade in a nongenomic manner.
There are two isoforms of PR, PRA and PRB. What are the different roles of PRA and PRB in the biological actions of progestin It has been reported that an imbalance in the native ratio of the two isoforms can lead to alterations in PR signaling (40) and mammary gland development (41). For example, PRB is required for progestin-dependent induction of vascular endothelial growth factor (42). Recently the presence of a specific membrane-bound PR in mature human spermatozoa was shown to regulate important sperm functions (43). PRB is also involved in the MPA-induced cyclin D1 expression via the ERK cascade (18) as well as involved in the MPA-induced cyclin D1 expression via the PI3K/Akt/NFB cascade, as detected in this study. Because blocking the ERK cascade with PD98059 had no effect on the MPA-induced Akt phosphorylation (data not shown), we believe that there is no cross-talk between the PI3K/Akt/NFB cascade and ERK cascade. Thus, because PRB induces both the PI3K/Akt/NFB and ERK cascade, there is a possibility that PRB localized in the membrane regulates cell proliferation in the mammary gland. However, the function of PRA remains unclear, and additional detailed examinations will be required to clarify it.
A network of interactions among PR signaling, type I IGF receptor (IGF-IR) signaling, and heregulin/ErbB-2 signaling was reported (45). Although MPA induces the phosphorylation of IGF-IR and ErbB-2, and IGF-IR directs ErbB-2 phosphorylation, blockage of either IGF-IR or ErbB-2 had no effect on the MPA-induced proliferation of breast cancer cells (44). Moreover, inhibition of breast cancer proliferation by blockage of IGF-IR involves inactivation of ErbB-2, PI3K/Akt, and MAPK signaling pathways but not modulation of PR activity, indicating that PR function remains unaffected by targeted blockage of IGF-IR (45). Thus, because cyclin D1 induction via the PI3K/Akt/NFB cascade seems to be necessary for the MPA-induced proliferation of breast cancer cells, it is important to investigate whether IGF-IR and/or ErbBs are involved in cyclin D1 induction via the PI3K/Akt/NFB cascade. We are currently examining this issue.
In the WHI study, although women on the CEE-only treatment arm did not show an increase in the risk for breast cancer (5), women on the CEE-MPA arm showed an increase in the risk for breast cancer (4). Are the mechanisms of estrogen- and progestin-induced cyclin D1 expression different The expression of cyclin D1 did not synergistically increase in the cells treated with both MPA and estrogen (data not shown), suggesting that the signaling cascade of MPA-induced cyclin D1 expression might be similar to that of estrogen-induced cyclin D1 expression. However, because it is known that the expression of PR is enhanced by the transcriptional activation by estrogen in the uterine endometrium (46) and mammary gland (47), estrogen priming may enhance the signaling cascade of PR. Further investigations will be necessary to explain the findings of the WHI study. Because the effect of MPA on the cell proliferation was examined here in human breast cancer cells, it is not yet possible to say whether progestin has a carcinogenetic effect on the normal breast epithelium. We are currently examining the effect of MPA on carcinogenesis using primary cultures of normal breast epithelium.
Footnotes
Abbreviations: CEE, Conjugated equine estrogens; FCS, fetal calf serum; h, human; IB, inhibitor of NFB; IGF-IR, type I IGF receptor; LUC, luciferase; MPA, medroxyprogesterone acetate; NFB, nuclear factor B; PI3K, phosphatidylinositol 3-kinase; PR, progesterone receptor; PRE, progesterone-responsive element; SDS, sodium dodecyl sulfate; TK, thymidine kinase.
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