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Breast Center (X.C., P.Z., W.D., S.O., A.V.L.), Baylor College of Medicine; and Department of Molecular Therapeutics (Y.L., G.B.M.), University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Address all correspondence and requests for reprints to: Adrian V. Lee, Ph.D., Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: avlee{at}breastcenter.tmc.edu.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Progesterone is also critically involved in the development of the mammary gland and breast cancer, and its effects are mostly mediated via the progesterone receptor (PR; Refs. 13, 14, 15). Mice lacking PR display incomplete mammary ductal branching and failure of lobular-alveolar development (16). Although PR expression is estrogen receptor (ER) dependent (17, 18), some breast cancer cell lines constitutively express high levels of PR independent of estrogens (19). Human PR normally exists in two isoforms (PR-A and PR-B) of 94 and 116 kDa, originating from two PR promoters (20).
Progesterone is considered differentiative in the uterus but proliferative in the normal mammary gland (14). However, progestin inhibition of breast cancer cell growth in tissue culture has been well documented (21, 22, 23). High doses of progestins have been used to treat estrogen-mediated mammary carcinomas, even though their antitumor mechanisms are not clear (14). Interestingly, progestins have been found to exert a biphasic regulation of breast cancer cell growth—accelerating cells through the first mitotic cell cycle, then arresting them in G1 of the second cycle. At this stage, the cell cycle progression machinery is poised to restart, as expression of growth factors and their receptors is increased by progestins (21, 22). Thus, it is proposed that progestins are neither inherently growth proliferative or inhibitive, but rather sensitize cells for growth factor and cytokine signals (24, 25).
The presence of PR in breast tumors is an important indicator of likely responsiveness to endocrine agents (13, 26). Approximately two thirds of breast cancers express the ER, some of which are ER positive (ER+)/PR negative (PR-). Their likelihood of response to endocrine therapy drops significantly compared with those that are ER+/PR+. It has also been reported that absence of PR in primary breast tumors is associated with secondary breast cancer in postmenopausal women (27), and absence of PR correlated significantly with a less differentiated phenotype of breast tumors (G1/G2 grading) and the presence of ErbB2/HER2/neu (28). Abnormal expression of ErbB2 and other growth factor receptors is normally associated with more aggressive tumors and a poorer patient prognosis (29, 30). So is there an intrinsic correlation between PR and intensity of growth factor action? We hypothesize that PR status may reflect growth factor function: low or absent PR expression indicates high IGF, epidermal growth factor (EGF), and heregulin (HRG) activities, and this correlation is independent of ER status. This assumption, combined with the fact that progesterone may inhibit breast tumor invasion, might explain why absence of PR is a marker of an aggressive tumor phenotype (27).
Recently, much effort has been directed to the study of the cross-talk between growth factors and ER signaling pathways in breast cancer cells (31), but how growth factors may interact with PR is less well defined. In this study, we have investigated the mechanisms for growth factor regulation of PR in breast cancer using IGF-I and MCF-7 cells because these cells are sensitive to the IGFs, have considerable PR levels, and possess intact PI3K, MAPK, and other common signaling pathways. We find that IGF-I dramatically down-regulates PR through a transcriptional mechanism involving the PI3K pathway, independent of ER activity. Our data provide the first evidence that activation of a growth factor signaling pathway can directly reduce PR levels, and may explain why PR-negative tumors, which possibly have highly active growth factor signaling, poorly respond to endocrine therapy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, A and B), which is physiologically relevant to the circulating concentrations of IGF-I in women (11). Much lower concentrations of IGF-I, 0.1 nM, also resulted in an apparent reduction of PR protein levels. From a time course experiment using 10 nM IGF-I, it was found that PR protein levels began to drop after 6 h of IGF-I treatment and continued to decrease with time, whereas the untreated control cells did not display visible down-regulation of PR (Fig. 1, C and D). After 24 h, the sharp fall in PR protein levels stopped, but levels remained suppressed for at least 72 h. We also observed similar results in other PR+ breast cancer cells such as T47D and ZR75 (data not shown). Hence, these data indicate that IGF-I lowers PR levels in breast cancer cells.
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). IGF-I pretreatment for 6 h caused a 70% reduction in R5020-induced luciferase activity. This was most probably due to the decrease of PR protein levels elicited by IGF-I, which by itself, at the same time point, had no detectable activation of the reporter activity over the unstimulated control. Hence, these data suggest that IGF-I represses PR transcriptional activity through down-regulation of PR in MCF-7 cells.
Because MCF-7 cells also respond well to other growth factors like EGF and HRG (although response to EGF is not as sensitive as the response to HRG and IGF-I), and these growth factors share similar signaling pathways, we subsequently examined the effect of EGF, HRG, and insulin on PR expression using immunoblotting. As a control, we treated cells with R5020 and found the characteristic upward protein mobility shift (due to phosphorylation) and decrease in PR levels (due to proteasomal degradation—see Fig. 2B) as reported previously (32). Not surprisingly, HRG and EGF at similar concentrations to that of IGF-I also markedly down-regulated PR (Fig. 2A), but insulin required 100-fold higher concentrations than IGF-I to achieve the same PR reduction. This suggests that the reduction of PR expression by IGF-I is a common effect shared by other growth factors in MCF-7 cells.
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, A and B). Thus, the mechanism by which IGF-I down-regulates PR in breast cancer cells may be different from the 26S proteasome degradation pathway. This was confirmed by the inability of lactacystin, a 26S proteasome inhibitor, to block the decrease of PR protein levels by IGF-I (Fig. 2B). To further investigate how IGF-I affects PR protein stability, we used the translation inhibitor cycloheximide. Western blot analysis demonstrated that, in contrast to noncycloheximide conditions, PR protein levels were not lowered by IGF-I treatment compared with non-IGF treatment under cycloheximide conditions (Fig. 2C, see 12-h and 24-h lanes). This indicates that IGF-I does not impede PR protein stability, which is consistent with the result from the proteasome inhibitor assay (Fig. 2B).
IGF-I Represses PR Transcription
To investigate whether IGF-I suppresses PR protein levels by inhibiting PR transcription, we conducted real-time quantitative RT-PCR (Q-PCR) to detect changes of PR mRNA concentrations in MCF-7 cells after IGF-I treatment. Because more than five PR mRNA transcripts with a wide range of sizes have been found in breast cancer cells and tumors, and it has not been clarified which codes for PR-A, PR-B, or both (20, 34), we chose primers and the fluorescent probe located near the 3' end of the PR coding region so as to detect all PR transcripts. We found that total PR mRNA transcript levels gradually decreased during the 24-h time period (Fig. 3A), which can probably be attributed to the fact that PR is ER dependent and estradiol is depleted in the serum-free medium (SFM) that was used in our cell stimulation experiments. However, compared with controls, PR mRNA levels dropped dramatically after 2 h of IGF-I treatment and continued to decrease with time, which was consistent with the change of PR protein levels in the IGF-I time course experiment (Fig. 1B). Interestingly, PR protein levels did not show a detectable drop at the 2-h time point, suggesting that the repression of PR mRNA levels by IGF-I occurred earlier than the reduction of PR protein levels. At any selected time point, PR mRNA levels under IGF-I-treated conditions were clearly lower than untreated controls. The decrease of PR mRNA levels by IGF-I was confirmed by Northern blotting using a probe at the 3' end of the PR coding region (data not shown). These results indicate that IGF-I represses PR mRNA levels in breast cancer cells.
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). However, in contrast to non-DRB conditions (Fig. 3A), PR mRNA levels under DRB conditions were not lowered by IGF-I compared with non-IGF-I treatment. The same result was confirmed by Northern blotting (data not shown). This suggests that IGF-I does not impair PR mRNA stability.
To confirm that IGF-I down-regulates PR through regulation of PR promoter activity, we transiently transfected a FLAG epitope-tagged PR-B cDNA and an HA epitope-tagged IRS-1 cDNA, both of which were driven by the cytomegalovirus (CMV) promoter, into MCF-7 cells. As expected, immunoblotting showed that IGF-I treatment did not reduce the exogenous PR protein levels that were generated from a heterologous promoter (CMV), whereas it did lower the exogenous IRS-1 levels and cause its mobility up-shift (Fig. 3C). As a control, the exogenous PR protein could still be regulated normally by R5020, i.e. PR protein levels were decreased and PR protein mobility was shifted upward. Furthermore, we stably transfected the PR-B cDNA into a specifically selected MCF-7 cell subline, which does not have detectable ER and PR but still responds to IGF-I (35). Similar to the transient transfection, IGF-I did not decrease PR-B levels in the stable transfectants but altered the endogenous IRS-1 levels and mobility (Fig. 3C). Then we transiently transfected a PRE-luciferase construct into these PR-B stable transfectants. Opposite to the data in regular MCF-7 cells, IGF-I could not inhibit the R5020-induced luciferase activity (data not shown).
In summary, these data show that IGF-I down-regulates PR mRNA transcription, and this is dependent upon the original PR promoter context.
IGF-I Regulates PR Expression through the PI3K/Akt/Mammalian Target of Rapamycin (mTOR) Pathway
The transmission of IGF signals in breast cancer cells involves several well-characterized signaling cascades downstream of IGF-I receptor (IGF-IR), including the PI3K/Akt and Ras/MAPK pathways (2). To identify the signaling pathway implicated in the IGF-I down-regulation of PR, we used a series of potent signaling inhibitors against Akt, MEK, mTOR, p38 MAPK, PI3K, protein kinase A (PKA), and protein kinase C (PKC). These inhibitors have been widely used in signal transduction research including our own previous studies (33). MCF-7 cells were first preincubated with these inhibitors and then stimulated with IGF-I in the presence of the inhibitors. Western blot analysis showed that blockade of Akt with 10 µM 1L-6-hydroxymethyl-chiro-inositol- (R)-2-O- methyl-3-O-octadecylcarbonate (HIMOC), of mTOR with 40 nM rapamycin, or of PI3K with 20 µM LY294002, rescued IGF-I down-regulated PR to the control level (Fig. 4A). In contrast, the MEK inhibitor U0126, the p38 MAPK inhibitor SB203580, the PKA inhibitor H-89, and the PKC inhibitor GF109203X did not prevent IGF-I reduction of PR in MCF-7 cells. These results were confirmed using other common inhibitors against the same target kinases such as the MEK inhibitor PD98059, the PI3K inhibitor wortmannin, and the PKC inhibitor GO6983 (data not shown). As PI3K, Akt, and mTOR form a sequential signaling cascade activated by the IGF-IR (36), the IGF-I effect on PR appears to be specifically elicited via the PI3K/Akt/mTOR pathway. Not surprisingly, the same signaling pathway was found to be involved in the EGF, HRG, and insulin down-regulation of PR (Fig. 4B). Because IGF-I represses PR at the transcription level, we confirmed by RT-PCR that inhibitors of PI3K or mTOR abolished the decrease of PR mRNA by IGF-I (Fig. 4C).
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), IGF-I down-regulates R5020-induced PR transcriptional activity. To assess the role of PI3K and mTOR in this event, we transiently transfected a PRE-luciferase construct into MCF-7 cells. Subsequent stimulation experiments using R5020, IGF-I, and the PI3K and mTOR inhibitors showed that the inhibitors impaired IGF-I reduction of R5020-induced luciferase activity (Fig. 4D). Noticeably, luciferase activity did not fully recover, unlike the PR protein levels (Fig. 4A). We speculate that the toxicity of these inhibitors to transfected cells and to the transcription machinery on the PRE promoter may contribute to their partial effect.
To confirm that the PI3K/Akt/mTOR pathway is involved in IGF-I down-regulation of PR, we used MCF-7 cells stably transfected with either a kinase-defective Akt (KD-Akt) containing a K179M substitution or a constitutively active Akt (myr-Akt) containing a myristoylation membrane-targeting sequence. Western blot analysis showed that overexpression of KD-Akt moderately elevated PR levels in MCF-7 cells in SFM, whereas overexpression of myr-Akt dramatically decreased PR levels (Fig. 5A). Interestingly, ER
protein levels were not different in the parental and transfected MCF-7 cells. As expected, mTOR phosphorylation was attenuated in KD-Akt cells but enhanced in myr-Akt cells (Fig. 5A). These data are consistent with Akt having a role in IGF-I down-regulation of PR in breast cancer cells.
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, 10 nM estradiol induced PR expression over the basal control levels in both KD-Akt and myr-Akt cells, suggesting that ER functions properly in both cell lines and that the effect of mutant Akt on PR is not mediated by influencing ER. Interestingly, LY294002 pretreatment displayed a stronger effect in blocking the IGF-I effect in KD-Akt transfectants than in myr-Akt transfectants, although rapamycin had a similar effect in both cells (Fig. 5B). This can be explained by the fact that Akt is downstream of PI3K and upstream of mTOR, and that myr-Akt activity is largely independent of growth factor and PI3K activation. Taken together, our data demonstrate that the PI3K/Akt/mTOR pathway uniquely mediates the alteration of PR expression elicited by IGF-I in breast cancer cells.
IGF-I Down-Regulation of PR Is Independent of ER Activity
It has been reported previously that IGF-I down-regulates ER levels in breast cancer cells (37, 38), which we also found (data not shown). Accordingly, this raised a question as to whether the IGF-I effect on PR was actually caused by the IGF-I effect on ER. This intriguing enigma prompted us to explore if there is a correlation between ER and PR expression regulated by IGF-I.
Initially, we had three pieces of evidence to suggest that the down-regulations of PR and ER are two independent events triggered by IGF-I. First, PR expression in T47D breast cancer cells, which is independent of ER (19), was also significantly reduced by IGF-I (data not shown). Second, as shown in Fig. 5, PR levels were markedly lower in myr-Akt stable transfectants than in KD-Akt cells, even though ER levels, and ER’s ability to induce PR, were similar in both cells. Third, PR levels were remarkably lower when MCF-7 cells were grown in dextran-charcoal-treated serum (DCS: estradiol and other steroid hormones removed, but growth factors maintained) medium than in SFM, whereas ER levels were constant in both media (data not shown). Thus, to further test this separation of effects, we performed the following experiments.
Previous reports have shown that IGF-I can cause ligand-independent activation of ER via alteration of ER phosphorylation status in breast cancer and other cells, and even in vivo (39, 40, 41). To test this in our experimental system, we transiently transfected an estrogen response element (ERE)-luciferase vector into MCF-7 cells, which were then stimulated with estradiol, IGF-I, or the antiestrogen ICI 182,780 (ICI). As shown in Fig. 6A, luciferase activity was significantly increased by estradiol or IGF-I and was further elevated by the two together, whereas ICI blocked the effect of both estradiol and IGF-I, suggesting that the IGF-I-induced luciferase activity is mediated by ER and accordingly that IGF-I can directly induce the transcriptional activity of ER. Surprisingly, Western blot analysis showed that estradiol and IGF-I cotreatment severely attenuated estradiol-induced PR levels, which dropped even lower than that of the unstimulated control (Fig. 6B). This suggests that IGF-I may directly repress PR expression, even while it activates ER, and that this repression is dominant over ER activity on PR. In contrast to PR, expression of cyclin D1, which is also ER dependent (42), was increased by estradiol, IGF-I, and the two together (43, 44). Thus, IGF-I has opposite effects on ER-inducible PR and ER-inducible cyclin D1 expression, even though it activates ER transcriptional activity.
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, estradiol or IGF-I treatment caused a 2- to 3-fold increase of the reporter activity over the nontreated control, whereas overexpression of myr-Akt or myr-PI3K led to a similar or higher increase of the luciferase activity in the absence of estradiol. This was due to the ligand-independent activation of ER by Akt and PI3K (PI3K utilizes both Akt-dependent and Akt-independent pathways) (45, 46). On the other hand, when we transiently cotransfected PRE-luciferase with myr-Akt or myr-PI3K into MCF-7 cells, either IGF-I or overexpression of myr-Akt or myr-PI3K inhibited the R5020-induced luciferase activity (Fig. 7B). Thus, myr-Akt and myr-PI3K can mimic the effect of IGF-I in suppressing PR activity through down-regulation of PR. Taken together, these data indicate that IGF-I regulates ER activity and ER-dependent PR levels in opposite ways, and that IGF-I down-regulation of PR expression is independent of ER (Fig. 8).
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DISCUSSION |
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). In this study, we found that EGF, HRG, and insulin can also sharply reduce PR levels. An obvious common feature between IGF-I and these growth factors is that they all can activate the PI3K/Akt signaling cascade in breast cancer cells, which explains why they all display the same effect on PR expression. One could speculate that other growth factors like PDGF, which also activates the PI3K/Akt pathway through its receptors, may likewise down-regulate PR in breast cancer cells that express PDGF receptors (47). But why does insulin require much higher doses to achieve the same effect as IGF-I even though MCF-7 cells have relatively high levels of insulin receptor (48)? We found that IGF-I is more potent than insulin at the same concentration (10 nM) to induce phosphorylation of IRS-1 and Akt, an indicator of activated PI3K/Akt signaling (Ref.33 and data not shown). Thus, high concentrations of insulin may be necessary for insulin to act through IGF-IR to decrease PR levels in MCF-7 cells because insulin has a relatively low affinity to IGF-IR (33, 49). Alternatively, high doses of insulin may simply enhance the insulin receptor-mediated PI3K/Akt signaling pathway.
It is a long-held rule of thumb that optimal estradiol stimulation of PR occurs in medium supplemented with 5% DCS (50). This may be attributed to the fact that DCS medium contains large amount of growth factors and consequently suppresses PR to barely detectable levels, whereas the basal level of PR is much higher in SFM. Hence, the fold-induction of PR with estradiol is seemingly more dramatic in DCS medium. This is perhaps also the reason behind the previous conflicting reports in which PR was used as a reporter gene monitoring ER activity in the study of IGF-I regulation of ER. Clayton et al. (37) showed that insulin and IGF-I considerably impaired the estradiol induction of PR mRNA in SFM, which is in line with our result. In another study that showed that IGF-I had a potentiating effect on the estradiol induction of PR mRNA (38), the cells were grown in DCS medium for 2 d. In this system, because PR transcription was already suppressed by growth factors, the observed IGF-I effect might actually indicate a change in PR mRNA and/or protein stability. Previously, Cho et al. (50) reported that IGF-I does not affect PR transcription but elevates PR protein levels in MCF-7 cells under SFM conditions. The discrepancy between their and our results may be due to the different cell culture conditions before IGF-I stimulation. In our study, before IGF-I treatment, cells were starved for 16 h after being switched to SFM from regular culture medium, whereas there was no delay of IGF-I treatment after the change to SFM from DCS medium in their studies.
Recently, evidence is emerging to support our conclusion that IGF-I and other growth factors inhibit PR expression. First, it was found that overexpression of ER in a specifically selected ER-/PR- MCF-7 breast cancer cell subline did not restore PR expression, although ER did restore cyclin D1, IRS-1, and IGF-IR levels (35). These cells were grown in DCS medium, which might completely suppress PR expression after long-term cell culture. Similarly, it was reported that in antiestrogen-resistant MCF-7 cells generated by continuous culture of the PR+ parental cells in antiestrogen-supplemented DCS medium, EGF receptor signaling was enhanced, whereas PR levels diminished (51). Interestingly, replacement of antiestrogen by estradiol failed to induce PR, whereas expression of other estrogen-responsive genes was significantly elevated. Conceivably, DCS medium plus enhanced EGF receptor signaling may permanently silence PR expression. In another interesting report, the mammary epithelial cells that retain stem/progenitor cell characteristics were found to lack PR (52). These cells rely on growth factors to continuously proliferate. Recently, in a study that confirmed that ER+/PR+ breast cancer patients respond better to hormonal therapy than ER+/PR- patients, Dowsett et al. (53) found that among ER+/PR- samples 25% are HER2+, compared with 10% HER2+ among ER+/PR+. Moreover, HER2+/PR- patients responded much worse than HER2-/PR+ in hormonal therapy, again suggesting that PR status may reflect HER2 signaling.
Our result indicates that the PI3K/Akt/mTOR pathway is responsible for the IGF-I down-regulation of PR. In a relevant report, Shen et al. (54) showed that, although overexpression of MEKK1, through p42 and p44 MAPK, increased R5020-induced PR activity, it alone could not down-regulate PR. Thus, PI3K/Akt and Ras/MAPK pathways may play distinct roles in regulating PR activity in breast cancer cells. However, we only observed IGF-I down-regulation of PR despite IGF-I’s ability to trigger both PI3K/Akt and Ras/MAPK pathways in MCF-7 and other breast cancer cells. This may be due to the finding that Ras can also bind to and activate PI3K (55), and the PI3K/Akt pathway is very potent in eliciting IGF-I signals in MCF-7 cells. Hence, the PI3K/Akt pathway is dominant in IGF-I regulation of PR. Interestingly, the PI3K/Akt/mTOR pathway is also involved in the IGF-I reduction of IRSs (33). The former event is mediated by a transcriptional mechanism, whereas the latter is via the 26S proteasome pathway for protein degradation. Different downstream proteins of mTOR may account for this seeming dilemma—although p70 S6 kinase is a typical target for mTOR, other kinases or signaling proteins may also function downstream of mTOR. For example, it has been shown that insulin regulation of IGFBP-1 gene expression is dependent on mTOR but independent of p70 S6 kinase activity (56). We hypothesize that mTOR may directly or indirectly activate a PR transcription repressor or inactivate a transcription activator to mediate the IGF-I effect on PR expression (Fig. 8).
The ER and PR status in breast cancer is highly correlated with the response to endocrine therapy. Key areas of study in breast cancer are those mechanisms that regulate ER and PR expression. The loss of PR gene expression has been attributed to loss of heterozygosity, loss of ER function, and methylation of a CpG island in the PR promoter (26). Our data provide implications for another theory, in which potent growth factor signaling, especially PI3K/Akt, may contribute to the PR down-regulation. This is reflected clinically with reduced PR levels in breast tumors with HER2 amplification (53). Because the IGF-I effect involves a transcriptional mechanism, whether IGF-I increases methylation of PR promoter and accordingly silences PR expression remains an intriguing question.
Low or absent PR in primary breast cancer is associated with faster disease progression and poor response to hormonal therapy. Our results suggest that low PR may be serving as an indicator of activated growth factor signaling in breast tumor cells, and therefore of an aggressive tumor phenotype and resistance against hormonal therapy.
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MATERIALS AND METHODS |
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Cell Culture and Plasmids
MCF-7 cells have been maintained in our laboratory for many years (57). Cells were routinely maintained in improved MEM zinc option (IMEM) supplemented with 5% fetal bovine serum, 2 mM glutamine, 50 IU/ml of penicillin, 50 µg/ml of streptomycin, and 10 µg/ml insulin. SFM consists of IMEM without phenol red plus 10 mM HEPES (pH 7.4), 1 µg/ml of transferrin, 1 µg/ml of fibronectin, 2 mM glutamine, 50 IU/ml of penicillin, 50 µg/ml of streptomycin, and trace elements (BioSource Technologies, Inc., Camarillo, CA). Cells were kept at 37 C in a humidified incubator with 5% CO2. The estrogen-responsive reporter plasmid ERE-tk-luc contains a single consensus ERE upstream of a minimal thymidine kinase (tk) promoter and the luciferase (luc) gene (40). The PRE-tk-luc was constructed in the same way. The expression vectors for constitutively active myr-Akt and myr-PI3K were described elsewhere (58), and were gifts from Dr. Thomas Franke and Dr. Anke Klippel. The murine myr-Akt and myr-PI3K (p110) each have a myristoylation signal at the N terminus to target the protein to the membrane.
Cell Stimulation and Lysis
Cells were plated at a density of 1.5 x 106 per 6-cm-diameter dish (Becton Dickinson and Co., Lincoln Park, NJ) and allowed to grow for 48 h. Then the medium was changed to SFM, and 16 h later, the cells were stimulated with 10 nM IGF-I, 20 nM EGF, 10 nM HRG, or insulin at 10, 100, or 103 nM for different periods of time. For experiments using inhibitors, cells were first preincubated separately with GF109203X (5 µM), H-89 (10 µM), lactacystin (10 µM), LY294002 (20 µM), a HIMOC (10 µM), rapamycin (40 nM), SB203580 (10 µM), and U0126 (5 µM) for 30 min before stimulation with EGF, HRG, IGF-I, and insulin in the presence of an inhibitor as described previously (33). For the translation inhibitor cycloheximide (10 µg/ml), the same procedure was followed. Control cells were incubated with a similar concentration of the vehicle dimethylsulfoxide alone. After stimulation, cells were washed twice with ice-cold PBS and then lysed in 200 µl of lysis buffer, which contained 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 2 mM EDTA, 100 mM NaCl, 10% glycerol, and a fresh protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN). Cells were left on ice for 30 min, and then the cell lysate was clarified by centrifugation at 14,000 x g for 15 min at 4 C and stored at -20 C. Protein concentration of the supernatant was measured by bicinchoninic acid assay in accordance with the manufacturer’s instruction manual (Pierce Chemical Co., Rockford, IL).
Immunoblotting
Total protein (40 µg) was resuspended in denaturing sample loading buffer (3% dithiothreitol; 0.1 M Tris-HCl, pH 6.8; 4% sodium dodecyl sulfate; 0.2% bromophenol blue; 20% glycerol), separated by 8% SDS-PAGE, and electrophoretically transferred to a nitrocellulose membrane overnight at 4 C. The remaining steps were all performed at room temperature. The membrane was blocked with PBS plus 0.05% Tween-20 (PBST) containing 5% nonfat milk for 1 h and followed by incubation with a 1:1000 dilution of anti-ER (Novocastra Laboratories, Newcastle upon Tyne, UK), anti-FLAG (Sigma), anti-HA (Covance Laboratories, Inc., Richmond, CA), anti-IRS-1 (Upstate Biotechnology, Lake Placid, NY), anti-PR (DAKO Corp., Carpinteria, CA), anti-ß-actin (Upstate Biotechnology), and anti-cyclin D1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies in blocking solution for 1–2 h. For phospho-mTOR detection, the membrane was first washed three times for 5 min each with PBST and then incubated with a 1:1000 dilution of anti-phospho-mTOR antibody (Cell Signaling, Beverly, MA) in PBST. Subsequently, the membrane was again washed three times for 5 min each with PBST and then incubated with a horseradish peroxidase-linked secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ) at a dilution of 1:4000 in blocking solution. After the membrane was washed three times for 5 min each with PBST, bands were visualized by enhanced chemiluminescence according to the manufacturer’s protocols (Pierce Chemical Co.).
RNA Blotting and RT-PCR
Cells were plated at a density of 5 x 106 per 10-cm-diameter dish and allowed to grow for 48 h. Then the medium was changed to SFM. After 16 h starvation, the cells were stimulated with IGF-I for an indicated time period. When the transcription inhibitor DRB (50 µM) and the signaling inhibitors LY294002 and rapamycin were used, cells were first preincubated with them for 30 min before IGF-I stimulation. Total RNA was prepared with RNeasy Midi kit (QIAGEN, Valencia, CA) according to the instruction manual. RNA integrity was checked by separation on a 1% agarose gel. For PCR of the 430-bp PR gene fragment, a pair of primers (5'-CAGTGGGCAGATGCTGTATTTTGC-3', 5'-GTGCAGCAATAACTTCAGACATC-3') was designed toward the 3' end of the PR coding region. Another pair of primers (5'-GGCTCTCCAGAACATCATCCCTGC-3', 5'-GGGTGTCGCTGTTGAAGTCAGAGG-3') was used in PCR of the 299 bp glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene fragment (59). In the RT-PCR experiment, total RNA (2 µg) was used to produce cDNA with Superscript II reverse transcriptase (Invitrogen) in a 20-µl volume. Then, 1 µl from the cDNA synthesis reaction was added to PCR mixture, and PCR amplification was performed with PR and GAPDH primers with an annealing temperature at 60 C and 30 cycles. Products were revealed by ethidium bromide staining under UV after agarose gel electrophoresis.
Q-PCR
Basically, reverse transcriptions of PR mRNA were performed in 96-well optical plates (PE Applied Biosystems, Foster City, CA) using Superscript II reverse transcriptase. All RNA samples were first treated with deoxyribonuclease I to eliminate residual genomic DNA. The reverse primer (5'-GGCTTAGGGCTTGGCTTTC-3') is at the 3' end of the PR coding region. Total RNA of 100 ng in a 10-µl reaction volume was added to each well. The plates were incubated at 50 C for 30 min followed by 10 min at 72 C. Then real-time quantitative TaqMan PCR of PR cDNAs was conducted using a PR-specific double fluorescence-labeled probe (5'-TCCCACAGCCATTGGGCGTTC-3') in an ABI PRISM 7700 Sequence Detector (PE Applied Biosystems). ROX was used as a reference dye. The PCR mixture also contains 300 nM each of the forward primer (5'-GAGCACTGGATGCTGTTGCT-3') and the reverse primer. The plates were incubated at 94 C for 1 min, followed by 40 cycles at 94 C for 12 sec and 60 C for 1 min. FAM was used as the fluorescent reporter coupled at the 5' end of the probe, whereas Block Hole Quencher was conjugated to the 3' end. Each experiment included three nontemplate controls to detect any template contamination. In addition, a control lacking reverse transcriptase was included for each sample to detect any residual genomic DNA. Standard curves for the quantification of PR and ß-actin mRNAs were generated using serial 10-fold dilutions from 108 to 102 copies of synthesized templates. Q-PCR was performed in triplicate of each sample. The obtained PR mRNA concentration was normalized by the ß-actin mRNA value.
Transfections
MCF-7 cells were grown for 48 h in IMEM + 5% FBS till 80% confluence before transfection. Stable transfectants of myr-Akt and KD-Akt, which contains K179M substitution, were gifts from Dr. Adriana Stoica (58). Using Lipofectamine (Invitrogen), a FLAG-PR-B expression vector pSG5-hPR1, which was a generous gift from Dr. K. Horwitz, was cotransfected with the neomycin resistant gene vector pcDNA3.1 (Invitrogen) in a 20:1 ratio (PR vector: neomycin vector). G418 of 600 µg/ml was used to select stable clones that were later verified by Western blot analysis with the PR antibody. All transient transfections were performed using Polyfect reagents (QIAGEN) according to the instruction protocols. For cotransfections with FLAG-PR-B and HA-IRS-1 constructs, 0.1 µg DNA of each plasmid was added to one well in six-well dishes. After 18 h, the culture medium was changed to SFM, and cells were incubated for 12 h before stimulation with 10 nM IGF-I and 10 nM R5020 for 24 h. For transfections with only ERE-tk-luc and PRE-tk-luc constructs, cells in six-well dishes were transfected with 0.2 µg DNA. After 5 h, the serum medium was switched to SFM, and cells were incubated for 6–8 h in the presence or absence of IGF-I. Then the cells were stimulated with 10 nM R5020 10 nM or 10 nM estradiol for 10–12 h. For the study of the inhibitors, cells were preincubated with LY294002 and rapamycin for 30 min before IGF-I treatment in the presence of the inhibitors. To examine the effect of myr-Akt and myr-PI3K on ER and PR activity, cells in six-well dishes were cotransfected with 0.2 µg reporter plasmids and 0.5 µg myr-Akt, myr-PI3K or empty pcDNA3 vector. Transfection time was extended to 16 h to allow sufficient time for exogenous Akt and PI3K expression. Then the serum medium was switched to SFM, and cells were stimulated with estradiol or R5020 for 12 h. Twenty nanograms of a ß-galactosidase expression vector pSV-ß-Gal (Promega Corp., Madison, WI) were cotransfected as an internal control. Luciferase and ß-galactosidase assays were performed using the Promega Corp. assay system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication September 10, 2002. Accepted for publication December 23, 2002.
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