Ligand- and Cell-Specific Effects of Signal Transduction Pathway Inhibitors on Progestin-Induced Vascular Endothelial Growth Factor Levels in Human Breast Cancer Cells

Jianbo Wu, Sandra Brandt and Salman M. Hyder

Dalton Cardiovascular Research Center and the Department of Biomedical Sciences, University of Missouri, Columbia, Missouri 65211

Address all correspondence and requests for reprints to: Dr. Salman M. Hyder, Dalton Cardiovascular Research Center, 134 Research Park Drive, University of Missouri-Columbia, Columbia, Missouri 65211. E-mail: hyders{at}missouri.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We evaluated the signaling pathways involved in regulating vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, in response to natural and synthetic progestins in breast cancer cells. Inhibition of the phosphoinositide-3'-kinase (PI3-kinase) signaling pathway or the specificity protein-1 (SP-1) transcription factor abolished both progesterone- and medroxyprogesterone acetate (MPA)-induced VEGF secretion from BT-474 and T47-DCO cells. Inhibitors of the MAPK kinase 1/2/MAPK and N-terminal jun kinase/MAPK signaling pathways blocked both progesterone- and MPA-induced VEGF secretion in BT-474 cells. However, these inhibitors blocked only progesterone-, but not MPA-induced VEGF secretion in T47-DCO cells. Inhibitors of PI3-kinase or SP-1 blocked both progesterone- and MPA-induced increases in VEGF mRNA levels in T47-DCO cells. The proximal SP-1 sites within the VEGF promoter were critical for progestin-dependent induction of VEGF. In contrast, MAPK inhibitors did not block the progesterone- or MPA-induced increases in VEGF mRNA in T47-DCO cells, suggesting that MAPK inhibitors decreased progesterone-induced VEGF secretion in T47-DCO cells by blocking posttranscriptional mechanisms. The MAPK kinase/ERK/MAPK-independent induction of VEGF mediated by MPA was associated with the PRB [progesterone receptor (PR) B] isoform of the PR in T47-DCO cells. None of the inhibitors tested reduced basal PR levels or abrogated PR-dependent gene expression from a reporter plasmid, indicating that loss of PR function cannot explain any of the observed effects. Because the PI3-kinase signaling pathway and SP-1 transcription factor play critical roles in progestin-dependent VEGF induction, these may be useful targets for developing antiangiogenic therapies to prevent progression of progestin-dependent human breast cancers.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANGIOGENESIS, THE FORMATION of new blood vessels, is essential for tumor growth, expansion, and metastasis (1, 2). Clinical studies have suggested that targeting angiogenesis is a useful approach for controlling the growth of breast tumors (3, 4, 5, 6, 7). Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic growth factors, and its effects on the proliferation, survival, and permeability of endothelial cells have been extensively studied (8, 9). VEGF165 and VEGF121 are the predominant isoforms detected in most tissues (8). Whereas both VEGF165 and VEGF121 are released from normal and tumor cells to initiate angiogenesis, VEGF189 remains bound to the cell matrix (8). Angiogenesis is a complex process that is regulated by both activators and inhibitors (10, 11). It has recently been shown that an angiogenic switch is triggered when the level of activators exceeds that of inhibitors, allowing angiogenesis and subsequent tumor growth to proceed (10, 12). VEGF regulates the angiogenic switch by elevating the angiogenic potential of tissues. Although the precise signals that increase VEGF levels and/or allow tumor cells to acquire the angiogenic switch have not been completely delineated, hypoxia, oncogenes, pH, and steroid hormones are examples of stimuli that are known to induce VEGF expression in tumor cells (13, 14).

Breast cancer is the second most common cause of cancer death in women in the United States. Considerable interest has focused on the possibility of inhibiting VEGF to prevent or control the growth of breast cancer (15, 16, 17, 18, 19). For this reason, the role of VEGF and angiogenesis in breast cancer has been extensively studied (7, 19). The proliferation of many breast cancer cells is controlled by the sex steroids estrogens and progesterone. Although the effects of these hormones on the proliferation of breast cancer cells has been well studied (20), little attention has been paid to the role of sex-steroid hormones and their receptors in controlling the process of angiogenesis for nourishment of tumor tissue (14, 21).

Recent evidence has indicated that there is an increased risk of breast cancer in women that consume a combined regimen of estrogen and progestin for hormone replacement therapy (HRT), as compared with those that take estrogen alone or a placebo (22, 23). This has renewed interest in the role of synthetic progestins in tumor growth. We previously reported that both natural and synthetic progestins used in oral contraceptives or HRT induce VEGF secretion in T47-D and BT-474 breast cancer cells through the progesterone receptor (PR), mainly the progesterone receptor B (PRB) isoform (24, 25, 26). The hormone-induced expression of VEGF is PR dependent (27) and occurs primarily in cells that lack the p53 tumor suppressor protein (26). Inhibition of VEGF has been shown to reduce the growth of breast tumors in animal models, as well as the proliferation of breast cancer cells in vitro (17, 28, 29, 30, 31). Thus, it may be possible to control the growth of breast cancer in humans by blocking the effects of VEGF in vivo.

We have demonstrated that the natural hormone progesterone as well as the synthetic progestin medroxyprogesterone acetate (MPA), which is extensively used for HRT, regulate VEGF in a subset of breast cancer cells (24, 25, 26). However, it is not known whether these progestins induce VEGF expression in tumor cells through common or distinct pathways. The present study was designed to delineate the intracellular signaling pathways involved in this process. Identification of the specific pathways involved may provide a critical first step toward developing therapies to control the effects of progestins on VEGF-mediated angiogenesis.

We performed experiments designed to determine whether progesterone and MPA utilize similar signaling pathways to induce PR-dependent expression of VEGF in T47-DCO and BT-474 cells. We evaluated the phosphoinositide-3'-kinase (PI3-kinase) signaling pathway, the MAPK signaling pathways, and a pathway that regulates the transcription factor specificity protein-1 (SP-1), which is known to be involved in VEGF synthesis and release (32, 33). We report that progestin-induced regulation of VEGF in breast cancer cells is predominantly controlled by the PI3-kinase pathway and the SP-1 transcription factor. In addition, the induction of VEGF in response to the natural and synthetic ligand is differentially sensitive to inhibition of the MAPK kinases (MEK1/2) and the N-terminal jun kinase (JNK) MAPK signaling pathways in a cell-dependent manner. Furthermore, we report the novel observation that the MAPK signaling pathways regulate progesterone-dependent VEGF synthesis and secretion at the posttranscriptional level in a subset of breast cancer cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inhibitors of Various Signal Transduction Pathways Regulate Progestin-Induced VEGF Secretion in a Ligand- and Cell Type-Dependent Manner in Breast Cancer Cells
We investigated the role of the PI3-kinase, MEK1/2/ERK/MAPK-, and JNK/MAPK signaling pathways and the SP-1 transcription factor on progestin-induced expression of VEGF in two different breast cancer cell lines, T47-DCO and BT-474 cells. These cells lines contain tumor suppressor p53 with mutations within the DNA binding domain of the protein (26, 34). Most other breast cancer cell lines containing wild-type p53 do not respond to progestin by increasing VEGF levels (24, 26). The inhibitors used included LY294002 (a specific inhibitor of PI3-kinase), U0126 (a specific inhibitor of MEK1/2), SP600125 (a specific inhibitor of JNK), and mithramycin, which inhibits the transcriptional activity of SP-1 (35, 36, 37).

Figure 1AGo shows that both progesterone and MPA increase VEGF levels in BT-474 cells. All four of the inhibitors tested, LY294002, U0126, SP600125, and mithramycin, blocked both the progesterone- and MPA-induced increases in VEGF secretion in the BT-474 breast cancer cells. The inhibitors themselves (in the absence of progesterone and MPA) also decreased basal levels of VEGF production in these cells, with the SP-1 inhibitor mithramycin having the greatest effect on basal VEGF levels.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1. Effects of Inhibiting the PI3-Kinase, MEK/ERK, and JNK Signaling Pathways and the SP-1 Transcription Factor on Progestin-Induced VEGF Secretion in Breast Cancer Cells

BT-474 (A) and T47-DCO cells (B) were pretreated 30 min with either dimethylsulfoxide [vehicle control (Con)], 20 µM LY294002 (LY), a PI3-K inhibitor, 20 µM U0126 (U), an MEK1/2 inhibitor, 25 µM SP600125 (S), a JNK inhibitor, or 50 nM mithramycin (M), an SP-1 inhibitor. Cells were then treated either with or without 10–8 M progesterone or 10–8 M MPA for 18 h in the continued presence of inhibitor (+inhibitor). One set of cells were treated with inhibitors alone without addition of any hormonal ligands (inhibitors alone). VEGF levels were measured by ELISA, as described in Materials and Methods. Data are expressed as average VEGF levels ± SEM of at least six determinations. {wedge}, P < 0.05 vs. control (Con). Asterisks represent VEGF induction statistically significant from that obtained with progesterone alone. Double asterisks indicate values that are significantly different from MPA alone (P < 0.05; two-tailed Student’s t test). C, The effects of the various inhibitors on PRA and PRB protein levels in BT-474 and T47-D cells, as determined by Western blotting. The concentrations and incubation times of the various inhibitors are the same as those used in panels A and B. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels are shown as a control.

 
We next evaluated the role of these signal transduction pathways on progestin-induced VEGF expression in T47-DCO cells. In contrast to the effects observed in BT-474 cells, the various inhibitors had differential effects on VEGF secretion in T47-DCO cells (Fig. 1BGo). All of the inhibitors blocked the effects of progesterone on VEGF secretion from the T47-DCO cells. However, only the PI3-kinase inhibitor (LY294002) and the SP-1 inhibitor (mithramycin) blocked MPA-induced VEGF secretion in this cell type (Fig. 1BGo). The MEK1/2 and JNK inhibitors had no effect on MPA-induced VEGF levels. SB203580, which specifically inhibits the phosphorylation and activation of the p38 MAPK, also had no effect on MPA-induced VEGF secretion (data not shown). These MAPK inhibitors also failed to block VEGF secretion when T47-DCO cells were stimulated with norgestrel, another synthetic progestin (data not shown). Both LY294002 and mithramycin also inhibited basal levels of VEGF secretion from the T47-DCO cells (Fig. 1BGo). These results suggest that signaling through the PI3-kinase and SP-1 pathways directly regulate secretion of VEGF from BT-474 and T47-DCO cells (Fig. 1Go, A and B).

To determine whether the inhibitors used in this study influenced PR levels, we incubated T47-DCO and BT-474 cells for 18 h in the presence or absence of these inhibitors and determined PR levels by subjecting whole cell extracts to Western blot analysis. As shown in Fig. 1CGo, although there was some variation in the overall levels of PR, PR levels remained stable during treatment with the various inhibitors tested in either cell line within the time frame used for these experiments.

The PRA and PRB Isoforms Differentially Inhibit Ligand-Dependent VEGF Secretion in Response to Inhibition of MEK1/2
We have previously shown that PRB appears to be the dominant receptor that controls VEGF synthesis and secretion in the T47-DCO cells (25). Because the MAPK inhibitors differentially inhibited progesterone- and MPA-induced increases in VEGF secretion in T47-DCO cells, we investigated whether this effect was specific for the individual PR isoforms. To do this, we used two different T47-DCO-derived breast cancer cell lines that were engineered to express equal levels of either only PRA (YA) or only PRB (YB) (38). These cell lines have also been shown to retain other (PR independent) signaling pathways that control VEGF secretion (25).

YB and YA cells were pretreated with LY294002, U0126, SP600125, or mithramycin before being exposed to progesterone, and the resulting VEGF levels were determined. As in the parental T47-DCO cells, LY294002 blocked progestin-dependent VEGF secretion from both YB and YA cells, indicating that the PI3-kinase pathway predominantly controls progestin-dependent induction of VEGF in T47-DCO breast cancer cells (Fig. 2Go, A and B). LY294002 also inhibited basal levels of VEGF secretion from both YA and YB cells. Thus, inhibition of the PI3-kinase signaling pathway blocked the progestin-dependent induction of VEGF irrespective of the ligand or receptor subtype present. In YB cells, the MEK1/2 inhibitor U0126 blocked progesterone-dependent VEGF secretion, but not MPA-dependent VEGF secretion (Fig. 2AGo). In YA cells, however, U0126 blocked the VEGF secretion induced by both progestin ligands (Fig. 2BGo). U0126 also blocked the basal level of VEGF secretion from the YA cells. In contrast, the JNK inhibitor SP600125 only partially blocked progesterone-induced VEGF secretion from YB and YA cells, and it did not inhibit the MPA-induced VEGF secretion from either cell type (Fig. 2Go, A and B). Similar results were obtained when the effects of these inhibitors were tested on norgestrel, another synthetic progestin (data not shown). It is important to note that the insensitivity of MPA-induced VEGF secretion to the MEK1/2 inhibitor U0126 seems specific to YB cells obtained initially from the T47-DCO cell type (38), as U0126 still abrogated MPA-dependent induction of VEGF in BT-474 cells, even when these cells were transiently transfected with a PRB expression vector (data not shown). Mithramycin, which inhibits the actions of the SP-1 transcription factors, completely abrogated progesterone- or MPA-induced VEGF induction in both YB and the YA cells (Fig. 2Go, A and B). Mithramycin also inhibited the basal level of VEGF secretion from the YB and YA cells.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2. Effects of Inhibiting the PI3-Kinase, MEK/ERK, and JNK Signaling Pathways and the SP-1 Transcription Factor on Progestin-Induced VEGF Synthesis in YB (A) and YA (B) Cells

Cells were treated with the various inhibitors and progestins and VEGF levels were measured exactly as described in Fig. 1Go. Data are expressed as average VEGF levels ± SEM. {wedge}, P < 0.05 vs. control. Asterisks represent induction values significantly different from progesterone alone. Double asterisks indicate values that are significantly different from MPA alone (P < 0.05; two-tailed Student’s t test).

 
Influence of Progesterone and MPA on Phosphorylation of Downstream Targets for PI3-Kinase Pathway (AKT) and ERK/MAPK Pathway (p44/42) in Breast Cancer Cells
Increasing evidence has shown that progestins mediate activation of the MAPK and PI3-kinase signaling pathways in several cell lines (39, 40, 41, 42). Because progesterone and MPA showed similar and differential response to various inhibitors for suppression of VEGF secretion in BT-474 and T47-D cells (Figs. 1Go and 2Go), we tested the ability of natural and synthetic progestins to activate the PI3-kinase and MEK/ERK signaling pathway in these cells by examining the phosphorylation status of their substrates AKT and p44/42. We first examined the effects of PI3-kinase and ERK pathway inhibitors in BT-474 cells. As shown in Fig. 3AGo, progesterone caused a rapid and transient activation of the ERK pathway (within 2 min of ligand exposure). At longer time points (>10 min), phospho-ERK (p-ERK) levels decreased to below control values. Progesterone also induced a rapid phosphorylation of Ser473-AKT (within 2 min). However, p-AKT levels then decreased at 10 min and once again became phosphorylated at 40 and 120 min (Fig. 3AGo). Similar results were obtained when BT-474 cells were stimulated with the synthetic progestin MPA (Fig. 3BGo). As shown in Fig. 3CGo, progesterone- and MPA-induced activation of ERK was blocked by both U0126 and by the progesterone antagonist RU-486. The effect of RU-486 indicates that the phosphorylation of ERK induced by progesterone and MPA is PR dependent. As shown in Fig. 3DGo, LY294002 blocked both progesterone- and MPA-induced phosphorylation of AKT in BT-474 cells.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3. Effects of Natural and Synthetic Progestins on Activation of the ERK and PI3-Kinase Signaling Pathways in BT-474 Cells

A, 10–8 M Progesterone or (B) 10–8 M MPA were added to BT-474 cells for the indicated times. Whole cell lysates were subjected to western blot analysis for phospho-ERK, total ERK, phospho-AKT (Ser 473), and total AKT, as shown. C and D, Confluent cells were pretreated 30 min with dimethylsulfoxide (DMSO) (C), 20 µM U0126 (U), 20 µM LY294002 (LY), or 10–6 M RU486 (RU), followed by treatment with 10–8 M progesterone (P) or 10–8 M MPA. Whole cell lysates were subjected to Western blot analysis for phospho-ERK, total ERK (C), or phospho-AKT (Ser 473), and total AKT levels (D).

 
We next evaluated the role of the ERK and PI3-kinase signaling pathways in the effects of progesterone and MPA on T47-DCO cells. As shown in Fig. 4AGo, progesterone rapidly but transiently induced phosphorylation of ERK. The maximal response was observed between 5 and 10 min after progesterone treatment and p-ERK levels diminished between 40 min and 120 min. Unlike progesterone, MPA did not induce any significant increase in phospho-ERK levels (Fig. 4BGo). However, p-ERK levels decreased below basal levels 40 and 120 min after exposure to MPA, similar to what was observed with progesterone. There were no changes in the total amount of ERK in response to either progesterone or MPA treatment.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 4. Effects of Natural and Synthetic Progestins on Activation of the ERK and PI3-Kinase Signaling Pathways in T47-DCO Cells

A, 10–8 M Progesterone or (B) 10–8 M MPA were added to T47-Dco cells for the indicated times. Whole cell lysates were then subjected to Western blot analysis for phospho-ERK, total ERK, phospho-AKT (Thr 308), phospho-AKT (Ser 473), and total AKT, as shown. C, Confluent cells were pretreated 30 min with dimethylsulfoxide (DMSO) (C), 20 µM U0126 (U), 20 µM LY294002 (LY), or 10–6 M RU486 (RU), followed by treatment with 10–8 M progesterone (P) or 10–8 M MPA, as indicated. Whole cell lysates were then subjected to Western blot analysis for phospho-ERK, total ERK, phospho-AKT (Ser 473), and total AKT, as shown.

 
We also examined the effects of progesterone and MPA on AKT, a downstream target of PI3-kinase. In contrast to the differential effects of progesterone and MPA on phosphorylation of ERK in T47-DCO cells, 40 min of exposure to either progesterone or MPA-induced phosphorylation of AKT at serine 473, but not at threonine 308 (Fig. 4Go, A and B). Progesterone-induced phosphorylation of ERK was completely blocked by both U0126 and RU-486 in T47-DCO cells (Fig. 4CGo). Each of these inhibitors also decreased basal levels of ERK phosphorylation. The effects of RU-486 suggest that progesterone-induced phosphorylation of ERK is PR-dependent and that the unliganded PR may play a role in maintaining basal levels of ERK activation. The PI3-kinase inhibitor LY294002 completely blocked progesterone- or MPA-induced phosphorylation of phospho-AKT at Ser473 (Fig. 4CGo).

Because the progestin-dependent induction of VEGF is primarily controlled by PRB (25), we next evaluated the roles of the ERK and PI3-kinase signaling pathways on the effects of these progestins in YB cells. As shown in Fig. 5Go, A and B, the effects of progesterone and MPA on ERK and AKT in YB cells was similar to that observed in the parental cells (Fig. 4Go, A and B). Progesterone rapidly stimulated the phosphorylation of ERK in YB cells (Fig. 5AGo), but MPA did not (Fig. 5BGo). However, both progesterone and MPA-induced phosphorylation of AKT at serine 473, indicating that the PI3-kinase signaling pathway was activated by both natural and synthetic progestin ligands (Fig. 5Go, A and B).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5. The ERK and PI3K-AKT Signaling Pathways Mediate Progestin-Induced VEGF Synthesis in YB Cells

A, 10–8 M Progesterone or (B) 10–8 M MPA were added to YB cells for the indicated times. Whole cell lysates were then subjected to Western blot analysis for phospho-ERK, total ERK, phospho-AKT (Thr 308), phospho-AKT (Ser 473), and total AKT, as shown. C, Confluent cells were pretreated 30 min with dimethylsulfoxide (DMSO) (C), 20 µM U0126 (U), 20 µM LY294002 (LY), or 10–6 M RU486 (RU), followed by treatment with 10–8 M progesterone (P) or 10–8 M MPA, as indicated. Whole cell lysates were then subjected to Western blot analysis for phospho-ERK, total ERK, phospho-AKT (Ser 473), and total AKT, as shown.

 
As in T47-DCO cells, progesterone-induced phosphorylation of ERK was completely blocked by U0126 in YB cells (Fig. 5CGo). However, RU-486 only partially blocked progesterone-induced phosphorylation of ERK in YB cells (Fig. 5CGo), unlike its effects in T47-DCO cells (Fig. 4CGo). The inability of RU-486 to completely block progesterone-induced ERK activation may explain the partial agonist effects of antihormones observed in the PRB cell line (43, 44, 45), as well as why RU-486 is a weak agonist for inducing transcriptional activity via a progesterone response element linked to a luciferase reporter plasmid (PRE-luc) in YB cells (43, 44).

To further characterize the roles of PI3-kinase and ERK in VEGF secretion, we treated T47-DCO and YB cells with the PI3-kinase inhibitor LY294002 before stimulation with progesterone. The effects of progesterone on the phosphorylation of AKT and ERK were then determined. As shown in Fig. 6Go, the PI3-kinase inhibitor LY294002 blocked progesterone-induced phosphorylation of ERK in both T47-DCO and in YB cells. As shown previously in Figs. 4CGo and 5CGo, LY294002 also blocks the phosphorylation of AKT in both T47-DCO and in YB cells. Thus, the findings presented in Fig. 6Go suggest that, like AKT, ERK is a downstream target for PI3-kinase mediated signaling events in T47-DCO cells.



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 6. Progesterone Activates ERK in T47-DCO and YB Cells

T47-DCO (top panel) and YB cells (bottom panel) were pretreated for 30 min with 20 µM LY294002 or vehicle, followed by treatment with 10–8 M progesterone or vehicle, as indicated. Whole cell lysates were then subjected to Western blot analysis for phospho-ERK and total ERK.

 
Progesterone and MPA Regulate the Phosphorylation of Cytosolic and Nuclear ERK in Breast Cancer Cells
The findings presented above indicate that progesterone and MPA have both similar and divergent effects on the MAPK activation in the BT-474 and T47-DCO cells (Figs. 1–6GoGoGoGoGoGo). Both ligands increased p-ERK levels in the whole cell extracts from BT-474 cells, but only progesterone increased p-ERK in T47-DCO cells. These results suggested that the point of convergence of these two hormones in T47-DCO cells is most likely at the level of MAPK activation because MPA fails to activate MAPK. However, other studies have shown that progesterone and MPA may differentially induce nuclear translocation of p-ERK in neuronal cells (46). To rule out this possibility as an explanation for the observed effects between progesterone and MPA in T47-DCO cells, we determined the effects of hormone exposure on p-ERK levels in cytosolic and nuclear extracts. As shown in Fig. 7Go, progesterone and MPA-regulated p-ERK levels in cytosolic and nuclear extracts in a pattern similar to what was observed in whole cell extracts (Figs. 3Go and 4Go). In BT-474 cells, both progesterone and MPA rapidly increased p-ERK in cytosolic and nuclear extracts (within 2 min), indicating that p-ERK was rapidly translocated to the nucleus (Fig. 7Go). In T47-DCO cells, progesterone treatment led to increased p-ERK within 2–5 min in both nuclear and cytosolic extracts; however, MPA did not increase p-ERK levels in either the cytosolic or nuclear fraction. In fact, MPA actually decreased p-ERK levels to below basal levels in both the cytosolic and nuclear fractions of T47-DCO cells, similar to what was observed in whole cell extracts (Fig. 4Go). These results indicate that the differential response of progesterone and MPA in the T47-DCO cells occurs at the level of activation of the MAPK pathway and is not dependent on the nuclear translocation of p-ERK.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 7. Activation of Cytosolic and Nuclear ERK in BT-474 and T47-D Cells with Progesterone and MPA

T47-DCO and BT-474 cells were treated with the indicated progestins. Nuclear and cytosolic fractions were prepared from cells at the indicated times (min) and were subjected to Western blot analysis for p-ERK and total ERK.

 
Influence of Signal Transduction Pathway Inhibitors on Progestin-Dependent Transcription of the Endogenous VEGF Gene
Because various inhibitors of specific signal transduction pathways differentially blocked progestin-induced VEGF secretion from T47-DCO cells, we next used real-time PCR to determine whether these inhibitors also differentially influenced expression of the VEGF gene. T47-DCO cells were treated with either LY294002 or U0126 for 30 min before a 6-h exposure to progesterone or MPA. The relative levels of VEGF mRNA were then determined by real-time PCR. As shown in Fig. 8Go, RU-486, LY294002, and mithramycin completely blocked progestin-dependent VEGF expression. However, neither U0126 and nor SB203580 blocked the progesterone or MPA-dependent increase in VEGF mRNA levels. This was an unexpected result because these MAPK inhibitors blocked the progesterone-dependent secretion of VEGF from T47-DCO cells under the same conditions.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 8. Effects of Inhibiting the PI3-Kinase, MEK/ERK, and JNK Signaling Pathways and the SP-1 Transcription Factor on Progestin-Induced VEGF mRNA levels in T47DCO Cells

Cells were pretreated 30 min in triplicate with either vehicle (C), 10–6 M RU-486 (RU), 20 µM LY294002 (LY), 20 µM U0126 (U), 25 µM SP600125 (S), or 50 nM mithramycin (M) followed by treatment with 10–8 M progesterone or 10–8 M MPA for 6 h in the continued presence of inhibitor. RNA was prepared and analyzed by real-time PCR as described in Materials and Methods. Data are expressed as average VEGF mRNA levels ± SEM, relative to control levels. Asterisk represents induction values statistically significant from P only samples, and double asterisk represent values significantly different from MPA only group (P < 0.05; two-tailed Student’s t test).

 
The Proximal SP-1 Recognition Sites Are Important for Progestin-Dependent Induction of the VEGF Promoter
The proximal SP-1 sites within the VEGF promoter have been shown to influence VEGF induction in response to many different signal transduction pathways, including their involvement in the induction of VEGF by estrogens (47, 48, 49). We considered the possibility that the proximal SP-1 sites may also be critically involved in the PR-induced expression of VEGF. As shown in Fig. 9AGo, both the full-length promoter (–2362/+956) and the truncated reporter plasmid (–135/+956), containing the four proximal SP-1 sites, were responsive to progesterone, similar to what has been reported previously in endometrial cells (50). However, progestin-dependent induction of VEGF was blocked when the SP-1 sites in the –135/+956 construct were either deleted (SacII construct, Fig. 9AGo) or when mithramycin was used to block SP-1 activity before the addition of progesterone (Fig. 9AGo). Furthermore, when all four SP-1 sites in the –135/+956 construct were mutated to prevent binding of the transcription factor to this region, progesterone failed to induce luciferase activity (Fig. 9BGo). These data suggest that the region within the VEGF promoter that includes the proximal SP-1 sites is critical for mediating progestin-dependent induction of VEGF in breast cancer cells.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 9. Progestin Induction of VEGF Promoter in the Presence and Absence of SP-1 Binding Sites

A, Full-length and truncated constructs of the VEGF promoter were transfected into the T47-Dco cells that were kept overnight in DMEM/F12 + 5% dextran-coated charcoal-stripped serum. Cells were then treated with mithramycin for 2 h, followed by incubation with 10 nM progesterone or 1 µM RU-486 for 18 h. {circ}, Four proximal Sp-1 sites. B, The four proximal SP-1 sites were mutated as shown in the upper panel. The wild-type and mutated constructs were transfected into T47-Dco cells and incubated for an additional 18 h in the presence of progesterone and/or RU-486. In the lower panel, {permzspch098} represent the mutated SP-1 sites. Asterisks represent induction values that are significantly different from controls (P < 0.05; two-tailed Student’s t test).

 
Inhibitors of the PI3-Kinase, MEK1/2, and JNK Signaling Pathways, and the SP-1 Transcription Factor, Do Not Inhibit PRE-Dependent Gene Expression in Response to Natural or Synthetic Progestins
Because LY294002 and mithramycin blocked the progesterone- or MPA-induced increases in VEGF mRNA levels in T47-DCO cells, we next asked whether these inhibitors had any effect on the ability of PR to induce PRE-dependent gene expression. T47-DCO and YB cells were transfected with the PRE-Luc reporter construct as described in Materials and Methods. The cells were pretreated for 30 min with the various inhibitors at the indicated concentrations, followed by treatment with either progesterone or MPA for 18 h. As shown in Fig. 10Go, A and B, none of the inhibitors that were tested completely blocked progestin-induced activation of the PRE-Luc construct. However, the SP-1 inhibitor consistently reduced progestin-induced activation of PRE-Luc. However, the MEK1/2 inhibitor consistently increased PRE-Luc activity in T47-Dco cells, but not in YB cells. Thus, the inhibition of progestin-induced expression of VEGF mRNA and VEGF secretion observed in response to inhibition of the PI3-kinase and SP-1 signaling pathways does not appear to be caused by direct effects of these signaling pathways on the PR protein or PR function.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 10. Effects of Inhibiting the PI3-Kinase, MEK/ERK, and JNK Signaling Pathways and the SP-1 Transcription Factor on the Transcriptional Activity of PR

T47Dco (A) and YB (B) cells were transiently transfected with 2 µg of the PRE-luciferase construct and then pretreated for 30 min with either vehicle (C), 20 µM LY294002 (LY), 20 µM U0126 (U), 25 µM SP600125 (S), or 50 nM mithramycin (M) followed by treatment with 10–8 M progesterone or 10–8 M MPA for 18 h in the continued presence of inhibitor. Cells were then lysed and luciferase activity was measured as described in Materials and Methods. Data are expressed as fold induction over control levels and represent the average of three determinations ± SEM. Asterisks represent induction values that were significantly different from controls (P < 0.05, two-tailed Student’s t test).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have previously shown that both natural and synthetic progestins increase VEGF expression and secretion from human breast cancer cells in a PR-dependent manner, and that this is mainly controlled by the PRB isoform (24, 25, 26, 27). PRB also appears to be a dominant regulator of gene expression for other progestin-responsive genes in endometrial cells (51). Synthetic progestins, particularly MPA, are commonly prescribed in therapy regimens to negate the untoward effects of estrogens on the uterus. The use of MPA by millions of women all over the world is associated with increased risk of breast cancer (22, 23). Because VEGF is a target for controlling the angiogenic switch as well as expansion and spread of tumors (10, 11), we investigated the signal transduction pathways used by progesterone and MPA that control VEGF production in T47-DCO and BT-474 breast cancer cells.

Our findings show that both natural and synthetic progestins induce VEGF expression and secretion primarily through the PI3-kinase signaling pathway and the SP-1 transcription factor. Furthermore, the proximal SP-1 binding sites on the VEGF promoter were critical for progestin-mediated VEGF induction. These proximal binding sites for SP-1 transcription factor have also been shown to play a role in mediating VEGF induction through various signal transduction pathways, including estrogen-dependent induction of VEGF (47, 48, 49). In BT-474 cells, both natural and synthetic progestins utilize the ERK and JNK MAPK signaling pathways in VEGF induction. However, only the natural hormone used these pathways in T47-DCO cells because MAPK inhibitors had no effect on MPA- or norgestrel-induced VEGF levels in these cells. This suggests that utilization of the MAPK signaling pathway by synthetic progestins occurs in a cell- and ligand-type-specific manner. To our knowledge, this is the first time that a potent angiogenic factor has been shown to be differentially controlled by synthetic progestins in different breast cancer cell types. The findings also suggest that MAPK inhibitors may not be suitable for effectively controlling progestin-dependent production of VEGF from certain breast cancer cells. Several previous reports have identified differences in regulation of natural vs. synthetic progestins, including differences in their ability to cause tumor cell proliferation (46, 52, 53, 54, 55). Other reports have also shown that natural and synthetic progestins differentially target the PI3-kinase and MAPK signaling pathways (42). The molecular basis for these differences between natural and synthetic progestins is uncertain. However, it is possible that differential metabolism of these progestins in various breast cancer cells may yield metabolites that do not effect the MAPK signaling pathway for increasing VEGF production from certain breast cancer cells. Further studies will be needed to evaluate the role of various progestin metabolites on VEGF induction in tumors.

In normal breast tissue, the ratio of PRA to PRB is close to one; however, this ratio can vary considerably in breast tumors (56, 57, 58). PRA and PRB differentially regulate progestin-responsive genes (25, 59, 60), and selective modulation of these receptor isoforms lead to characteristic reproductive abnormalities (61, 62). To determine whether the specific PR isoforms could differentially influence the activity of natural and synthetic progestins, we used T47-DCO-derived cells that express only PRA or PRB (38). We found that the MEK1/2 inhibitor U0126 blocked both progesterone- and MPA-induced secretion of VEGF in T47-DCO cells expressing only the PRA isoform. However, U0126 blocked progesterone, but not MPA-induced secretion of VEGF in cells that expressed only PRB. It is possible that in cells expressing only PRB, induction of a PRB-specific gene may lead to the metabolism of the synthetic progestin and thereby allow an alternative pathway to induce VEGF expression. A similar effect has been documented by Horwitz et al. and is believed to promote carcinogenicity with tamoxifen in a receptor-dependent manner (63). Further study will be necessary to understand why MPA-induced VEGF expression is independent of the ERK signaling pathway in YB cells. When PRB was overexpressed in BT-474 cells by transient transfection, progesterone- and MPA-induced VEGF expression remained sensitive to the MEK1/2 inhibitor U0126 (Fig. 1AGo and data not shown). Thus, the inability of U0126 to prevent MPA-induced VEGF production in YB cells seems to be cell type dependent. However, it is possible that transient transfections did not lead to PRB expression levels comparable to what are found in the T47-DCO cells. Also, unlike YB cells, the BT-474 cells transiently transfected with PRB also express PRA, and this may override any PRB-dependent effects that allow the ERK/MAPK pathway to be bypassed in BT-474 cells. It will be useful to isolate BT-474 clones that lack the PR, as has been done for T47-DCO cells (38). The individual PRA and PRB isoforms can then be reintroduced into the modified BT-474 cells to reach the levels similar to those present in the T47DCO cells. One could then determine whether high levels of PRB lead to MPA-dependent VEGF induction that is independent of the MAPK pathway. It is also important to note that although MPA-induced VEGF secretion could be blocked by inhibitors of MEK1/2/MAPK in the absence of PRB, MPA-induced VEGF secretion remained independent of the JNK/MAPK signaling pathway in YB, YA, and parental T47-DCO cells. Thus, MPA uses a pathway that is distinct from the one used by the natural hormone progesterone to mediate VEGF induction in T47-DCO cells. Interestingly, the JNK/MAPK signaling pathway is involved in VEGF induction in BT-474 cells because inhibition of JNK/MAPK blocked both progesterone-and MPA-induced VEGF expression in this cell type. Such heterogeneity among cells within a tumor may limit the effectiveness of drugs that target only the ERK signaling pathway to reduce progestin-mediated VEGF induction because there may be a continuous supply of an angiogenic growth factor under these conditions.

The ability of progesterone or MPA to regulate VEGF in BT-474 and T47-DCO cells was correlated with the ability of the ligand to induce activation of the PI3-kinase or the ERK signaling pathways. In BT-474 cells, both the PI3-kinase and the ERK signaling pathways were activated by progesterone and MPA treatment and this correlated with the ability of inhibitors of these pathways to block progesterone-or MPA-induced VEGF expression. However, the natural and synthetic progestins did not exert the same effects in T47-DCO and YB cells. As in BT-474 cells, progesterone activated both the ERK and the PI3-kinase pathways in T47-DCO and YB cells. However, MPA activated only the PI3-kinase pathway in T47-DCO and YB cells. Similar effects were observed in response to norgestrel, another synthetic ligand (data not shown), indicating that the structure of the natural ligand is important for initiating the effects of MAPK. Clearly, more extensive studies with various progestins will be required to better understand the role of ligand structure and metabolism and how these factors may control various signal transduction pathways in breast cancer cells. Nevertheless, it is intriguing that different ligands can regulate VEGF via specific signal transduction pathways in specific cell types. In addition to regulating VEGF, the MAPK signaling pathway is also known to regulate genes or proteins involved in cellular proliferation. In such situations, progestin-dependent VEGF induction would be predicted to lead to increased angiogenesis associated with increased cellular proliferation. This may be related to the finding that the use of synthetic progestins can lead to increased risk of breast cancer (22, 23).

To our knowledge, this is the first time that different signal transduction pathways have been shown to be used by natural vs. synthetic progestins for the induction of an angiogenic growth factor associated with tumor growth. This may represent the molecular basis for the different pathologies reported in progesterone- vs. MPA-induced breast tumors in mice (64, 65) or for the different potencies exhibited by natural and synthetic progestins for inducing proliferation of breast cancer cells in vitro (55, 66).

When RU-486 was used to block the PR, progestin-induced phosphorylation of ERK/MAPK was abolished, indicating that the PR plays a role in this process. This observation also raises the interesting possibility that the PR may possess intrinsic protein kinase activity and phosphorylate the p44/42 proteins directly, although, this remains to be shown. Protein kinase activity has been previously reported to be associated with the PR (67). It is also known that the PR can interact with src and the ER to regulate the MAPK pathway (68).

The progesterone-induced activation of the ERK/MAPK pathway was blocked by the PI3-kinase inhibitor LY294002 in T47-DCO cells, indicating that PI3-kinase lies upstream of the ERK/MAPK pathway in these cells and that there is cross talk between these pathways during regulation of VEGF gene expression. However, no such cross talk was observed in the effects of MPA on VEGF. Thus, although the natural hormone utilizes both the PI3-kinase and ERK signaling pathways, the ERK pathway is bypassed by synthetic progestins in certain cells, such as T47-DCO cells. Further investigation will be required to determine whether downstream targets of AKT are shared or distinct in the effects of natural and synthetic progestins on VEGF induction in breast cancer cells.

One of the most interesting findings in this investigation was that inhibition of progestin-mediated increases in VEGF secretion in T47-DCO cells by certain signal transduction inhibitors were not accompanied by loss of expression of the VEGF gene. Although inhibitors of the PI3-kinase signaling pathway and the SP-1 transcription factor abrogated VEGF mRNA expression and secretion from the breast cancer cells, irrespective of the ligand used, the MAPK inhibitors (SB203580 and U0126) blocked progesterone-induced VEGF secretion from these cells but failed to block increases in VEGF mRNA levels. This indicates a novel role for the MAPK signaling pathways in the posttranscriptional regulation of progesterone-induced VEGF production. Such regulation may occur at the level of transporting VEGF mRNA from the nucleus into the cytoplasm, at the level of mRNA stability, translation of mRNA into protein, or during the secretion of VEGF from cells. It has been shown that the MAPK signaling pathway is involved in the posttranscriptional control of proteins (69, 70). Further experiments will clearly be necessary to understand the precise role of MAPK inhibitors on ligand-dependent VEGF secretion in T47-DCO cells.

Interestingly, none of the signal transduction pathway inhibitors that we evaluated dramatically influenced the transcriptional potential of the PR, as determined by analysis of PR-dependent induction of PRE-Luc reporter plasmid. However, we noticed that the MEK1/2 inhibitor consistently increased the induction of PRE-luciferase activity in T47-DCO cells, but not in YB cells. This indicates that perhaps PRB is modifying certain inhibitors. The loss of these inhibitors could lead to the induction of luciferase activity in the presence of U0126. Alternatively, it is possible that coactivators/repressors or receptors are selectively being affected to enhance luciferase activity. It is also possible that in the presence of PRA, T47-DCO cells synthesize certain inhibitors of PRE-luc activity, possibly via the PR. Alternatively, PRA could control the activity of PRB, which leads to enhanced activation of luciferase activity. This suggests that the loss of YA in T47-Dco cells selectively allows YB cells to control a cellular component that either activates the PR or potentially stabilizes luciferase mRNA. These results indicate that although the various inhibitors showed slight differences in their abilities to activate PRE-Luc, none of them completely inhibited PRE-Luc, as does the receptor antagonist RU-486. These inhibitors did not alter PR levels in breast cancer cells, but it is possible that they could modify other cofactors associated with the PR. Our findings suggest that when progestin-mediated VEGF production is blocked by the presence of these inhibitors, other posttranscriptional (non-PRE) events must be involved or that other transcription factors (or coactivators/corepressors) are altered and unable to cooperate with the PR at the level of the VEGF gene.

One implication of the current findings is that the induction of VEGF, a potent angiogenic factor, may be involved in the increased risk of breast cancer observed in a subset of women who are prescribed progestins for HRT (22, 23). The ingestion of progestins may provide the angiogenic switch in preexisting lesions or tumors, and the increased VEGF may facilitate tumor growth. The PI3-kinase pathway and the SP-1 transcription factor appear to play important roles in progestin-mediated VEGF induction in tumor cells. It is important to mention that breast cancer cells could metabolize progestins to compounds that may trigger a growth response (71, 72, 73), which may also increase VEGF production. The synthetic progestins are less easily metabolized than the natural compound, which has a shorter half-life in tumor cells (71, 72, 73). Thus, some of the differences we have observed between BT-474 cells and T47-DCO cells with respect to the activation of signal-transduction pathways could result from differential progestin metabolism in these cells. Further studies will be needed to characterize the metabolism of progestins; nevertheless, this does not undermine our observation that breast cancer cells differ in their ability to respond to progestins and differentially activate pathways that could lead to the production of a potent angiogenic factor. Based on our observation that the PI3-kinase signaling pathway and the SP-1 transcription factor appear to be critical for VEGF induction in breast cancer cells, it appears that targeted therapy against PI3-kinase, SP-1, and/or PR may provide an effective antiangiogenic approach to curtail progression of progestin-dependent breast cancer by blocking the secretion of VEGF from breast tumor cells.

In conclusion, our study provides evidence that the PI3-kinase pathway and functional SP-1 protein predominantly control progestin-dependent VEGF induction in response to both the natural hormone progesterone and synthetic ligands in breast cancer cells. In contrast, the utilization of the MAPK pathway for progestin-dependent VEGF induction is ligand and cell type dependent. Thus, blocking the PI3-kinase signaling pathway or the SP-1 transcription factor and/or blocking PR with antiprogestins, could prove effective for suppressing progestin-induced proangiogenic signals that can provide the angiogenic switch for tumor progression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Progesterone (4-pregnene-3,20-dione), medroxy progesterone acetate (MPA; 17{alpha}-hydroxy-6{alpha}-methyl-4-pregnene-3,20-dione17-acetate), RU-486, mithramycin, and LY 294002 were purchased from Sigma (St. Louis, MO). U0126 and SB203580 were purchased from Calbiochem (La Jolla, CA). The inhibitors and concentrations used were chosen based on previous publications (35, 36, 37), and these concentrations represent values greater than their ED50 values.

Cell Culture
The PR-positive T47-DCO breast cancer cell line, isolation of T47-DCO-Y, its PR-negative clonal derivative, and construction of PR-positive YA and YB cells have been described previously (38). All cell lines were grown in phenol red-free DMEM/F12 (Invitrogen Life Technologies, Carlsbad, CA), supplemented with 5% fetal calf serum (FCS, JRH Bioscience, Lanexa, KS). Cells were routinely cultured in 100-mm dishes and incubated at 37 C with 5% CO2 in a humidified environment. The YA and YB cells were grown in media containing 200 µg/ml G418 (Sigma, St. Louis, MO) to select for stable expression of PRA and PRB. BT-474 cells were obtained from ATCC (Manassas, VA). The various inhibitors were added 30 min before the addition of progestin at the indicated concentrations.

VEGF ELISA
VEGF was measured with a Quantikine ELISA kit from R&D Diagnostics (Minneapolis, MN) according to the manufacturer’s recommended protocol. VEGF levels were normalized to total cellular protein in each dish using BCA protein assay kit (Pierce, Rockford, IL). Human recombinant VEGF165 was used as a standard and was provided by the manufacturer’s of the VEGF ELISA kit. Data were analyzed using a two-tailed Student’s t test. P values < 0.05 were considered to be statistically significant. Inter- and intraassay coefficients of variance were 5.0–8.5% and 3.5–6.5%, respectively, according to the manufacturer.

Plasmids, Transfections, and Luciferase Assays
To construct the luciferase reporter, the PvuII-SmaI fragment was excised from pPRE/GRE.E1b.CAT (26) and ligated into the SmaI site of pGL3Basic (Promega, Madison, WI). pPRE/GRE.E1b has two copies of the consensus PRE linked to the TATA element from E1b (kindly provided by Dr. Zafar Nawaz, Creighton University, Omaha, NE).

The promoter region of the human VEGF gene (–2362 to +956 relative to the transcription start site) and various deletion constructs were kindly provided by Dr. Lee Ellis (Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX) (74). The regulatory sequences were fused to the luciferase reporter gene in pGL3-basic vector (Promega, Madison, WI) to generate pVp-ecor. The proximal SP-1 sites in the human VEGF gene (ApaI construct, –135/+956) were mutated by site-directed mutagenesis at Top Gene Technologies (Montreal, Quebec, Canada) with bases that are known to abolish SP-1 binding to the gene (75, 76). The resulting construct was sequenced and the mutated bases are shown in Fig. 9BGo.

T47-DCO and variant cells were grown in DMEM/F12 supplemented with 10% FCS and plated at 3 x 105/well in Falcon six-well dishes in 5% dextran-coated charcoal-stripped serum 24 h before transfection. Cells were transfected with the indicated plasmids using Superfect reagent (QIAGEN, Valencia, CA) according to the manufacturer’s guidelines. Cells were washed with PBS and incubated in DMEM/F12 supplemented with 5% FCS in the presence of various hormones, as indicated. Cells were lysed after 20 h, and luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI) and a Sirius luminometer (Berthold Detection Systems, Pforzheim, Germany). All experiments were performed in triplicate and repeated at least twice. Data were normalized to Renilla luciferase (pRL-CMV plasmid, Promega E2261) activity and expressed as fold increase relative to control levels.

Immunoblotting
Whole cells lysates were prepared in RIPA buffer, as described previously (26). The cytosolic and nuclear extracts were obtained using NE-PER nuclear and cytoplasmic extraction reagents (Pierce). Equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. After blocking, the membranes were incubated with phosphospecific and nonphosphospecific antibodies directed against p44/p42 MAPK(Thr202/Tyr204), p38 MAPK(Thr180/Tyr182), AKT(Ser473) (Cell Signaling, Beverly, MA) and AKT(Thr308) (Upstate, Lake Placid, NY). The secondary antibody was an antirabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), diluted 1:2000 and incubated with the membrane for 1 h. Protein bands were detected using enhanced chemiluminescence plus detection system according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Arlington Heights, IL).

Quantitative Real-time PCR
Total RNA was extracted from cells using the Ultraspec RNA Isolation System (Biotex Laboratories, Inc., Houston, TX). RNA was pretreated with deoxyribonuclease I (Invitrogen Life Technologies), and SuperScript (Invitrogen Life Technologies) was used to synthesize cDNA, according to the manufacturer’s recommended conditions. The VEGF primers and probe were designed by Lark Technologies, Inc. (Houston, TX). These primers recognize mRNA derived from all VEGF isoforms. Real-time quantitative PCR analysis was performed using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Each sample was analyzed in duplicate with ribosomal 18S RNA was used as an internal control. The sequences of primers and probes used in both assays were proprietary. After amplification, the relative differences in amounts of RNA were calculated based on the 2{Delta} {Delta}CT method (Applied Biosystems Prism Sequence Detection System User Bulletin #2).


    ACKNOWLEDGMENTS
 
We are grateful to Dr. Zafar Nawaz from Creighton University (Omaha, NE) for the provision of PRE-Luc plasmid, to Dr. Kathryn Horwitz and Jennifer Richer from the University of Colorado (Denver, CO) for the T47-Dco and its variants, and to Dr. Lee Ellis from the M. D. Anderson Cancer Center (Houston, TX) for the provision of human VEGF promoter constructs.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grant CA-86916. S.M.H. is the Zalk Missouri Professor of Tumor Angiogenesis.

First Published Online November 4, 2004

Abbreviations: ER, Estrogen receptor; FCS, fetal calf serum; MEK, MAPK kinase; HRT, hormone replacement therapy; JNK, N-terminal jun kinase; MPA, medroxy progesterone acetate; PI3-kinase, phosphoinositide-3'-kinase; PR, progesterone receptor; PRB, progesterone receptor B; PRE, progesterone response element; SP-1, specificity protein-1; VEGF, vascular endothelial growth factor.

Received for publication June 23, 2004. Accepted for publication October 24, 2004.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Folkman J 1995 Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1:27–31[Medline]
  2. Ferrara N 2002 Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin Oncol 29:10–14
  3. Borgstrom P, Gold DP, Hillan KJ, Ferrara N 1999 Importance of VEGF for breast cancer angiogenesis in vivo: implications from intravital microscopy of combination treatments with an anti-VEGF neutralizing monoclonal antibody and doxorubicin. Anticancer Res 19:4203–4214[Medline]
  4. Moore MR 2004 A rationale for inhibiting progesterone-related pathways to combat breast cancer. Curr Cancer Drug Targets 4:183–189[Medline]
  5. Atiqur RM, Toi M 2003 Anti-angiogenic therapy in breast cancer. Biomed Pharmacother 57:463–470[CrossRef][Medline]
  6. Gasparini G 2000 Prognostic value of vascular endothelial growth factor in breast cancer. Oncologist 5(Suppl 1):37–44
  7. Locopo N, Fanelli M, Gasparini G 1998 Clinical significance of angiogenic factors in breast cancer. Breast Cancer Res Treat 52:159–173[CrossRef][Medline]
  8. Ferrara N 2001 Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol 280:C1358–C1366
  9. Ferrara N 1999 Molecular and biological properties of vascular endothelial growth factor. J Mol Med 77:527–543[CrossRef][Medline]
  10. Hanahan D, Christofori G, Naik P, Arbeit J 1996 Transgenic mouse models of tumour angiogenesis: the angiogenic switch, its molecular controls, and prospects for preclinical therapeutic models. Eur J Cancer 32A:2386–2393
  11. Folkman J, Hanahan D 1991 Switch to the angiogenic phenotype during tumorigenesis. Princess Takamatsu Symp 22:339–347[Medline]
  12. Folkman J, Watson K, Ingber D, Hanahan D 1989 Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 339:58–61[CrossRef][Medline]
  13. Claffey KP, Robinson GS 1996 Regulation of VEGF/VPF expression in tumor cells: consequences for tumor growth and metastasis. Cancer Metastasis Rev 15:165–176[Medline]
  14. Hyder SM, Stancel GM 1999 Regulation of angiogenic growth factors in the female reproductive tract by estrogens and progestins. Mol Endocrinol 13:806–811[Free Full Text]
  15. Sartor CI 2002 Molecular targets as therapeutic strategies in the management of breast cancer. Semin Radiat Oncol 12:341–351[CrossRef][Medline]
  16. Gasparini G 1997 Antiangiogenic drugs as a novel anticancer therapeutic strategy. Which are the more promising agents? What are the clinical developments and indications? Crit Rev Oncol Hematol 26:147–162[Medline]
  17. Zhang W, Ran S, Sambade M, Huang X, Thorpe PE 2002 A monoclonal antibody that blocks VEGF binding to VEGFR2 (KDR/Flk-1) inhibits vascular expression of Flk-1 and tumor growth in an orthotopic human breast cancer model. Angiogenesis 5:35–44[CrossRef][Medline]
  18. Longo R, Sarmiento R, Fanelli M, Capaccetti B, Gattuso D, Gasparini G 2002 Anti-angiogenic therapy: rationale, challenges and clinical studies. Angiogenesis 5:237–256[CrossRef][Medline]
  19. Gasparini G, Toi M, Gion M, Verderio P, Dittadi R, Hanatani M, Matsubara I, Vinante O, Bonoldi E, Boracchi P, Gatti C, Suzuki H, Tominaga T 1997 Prognostic significance of vascular endothelial growth factor protein in node-negative breast carcinoma. J Natl Cancer Inst 89:139–147[Abstract/Free Full Text]
  20. Pasqualini JR, ed. 2000 Breast cancer: prognosis, treatment and prevention. New York: Marcel Dekker
  21. Hyder SM, Stancel GM 2000 Regulation of VEGF in the reproductive tract by sex-steroid hormones. Histol Histopathol 15:325–334[Medline]
  22. Writing Group for the Women’s Health Initiative Investigators 2002 Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 288:321–333[Abstract/Free Full Text]
  23. Ross RK, Paganini-Hill A, Wan PC, Pike MC 2000 Effect of hormone replacement therapy on breast cancer risk: estrogen versus estrogen plus progestin. J Natl Cancer Inst 92:328–332[Abstract/Free Full Text]
  24. Hyder SM, Murthy L, Stancel GM 1998 Progestin regulation of vascular endothelial growth factor in human breast cancer cells. Cancer Res 58:392–395[Abstract]
  25. Wu J, Richer J, Horwitz KB, Hyder SM 2004 Progestin-dependent induction of vascular endothelial growth factor in human breast cancer cells: preferential regulation by progesterone receptor B. Cancer Res 64:2238–2244[Abstract/Free Full Text]
  26. Liang Y, Wu J, Stancel GM, Hyder SM, p53-Dependent inhibition of progestin-induced VEGF expression in human breast cancer cells. J Steroid Biochem Mol Biol, in press
  27. Hyder SM, Chiappetta C, Stancel GM 2001 Pharmacological and endogenous progestins induce vascular endothelial growth factor expression in human breast cancer cells. Int J Cancer 92:469–473[CrossRef][Medline]
  28. Bogin L, Degani H 2002 Hormonal regulation of VEGF in orthotopic MCF7 human breast cancer. Cancer Res 62:1948–1951[Abstract/Free Full Text]
  29. Shao ZM, Shen ZZ, Liu CH, Sartippour MR, Go VL, Heber D, Nguyen M 2002 Curcumin exerts multiple suppressive effects on human breast carcinoma cells. Int J Cancer 98:234–240[CrossRef][Medline]
  30. Im SA, Kim JS, Gomez-Manzano C, Fueyo J, Liu TJ, Cho MS, Seong CM, Lee SN, Hong YK, Yung WK 2001 Inhibition of breast cancer growth in vivo by antiangiogenesis gene therapy with adenovirus-mediated antisense-VEGF. Br J Cancer 84:1252–1257[CrossRef][Medline]
  31. Saaristo A, Karpanen T, Alitalo K 2000 Mechanisms of angiogenesis and their use in the inhibition of tumor growth and metastasis. Oncogene 19:6122–6129[CrossRef][Medline]
  32. Pal S, Claffey KP, Cohen HT, Mukhopadhyay D 1998 Activation of Sp1-mediated vascular permeability factor/vascular endothelial growth factor transcription requires specific interaction with protein kinase C{zeta}. J Biol Chem 273:26277–26280[Abstract/Free Full Text]
  33. Milanini-Mongiat J, Pouyssegur J, Pages G 2002 Identification of two Sp1 phosphorylation sites for p42/p44 mitogen-activated protein kinases: their implication in vascular endothelial growth factor gene transcription. J Biol Chem 277:20631–20639[Abstract/Free Full Text]
  34. O’Connor PM, Jackman J, Bae I, Myers TG, Fan S, Mutoh M, Scudiero, DA, Monks A, Sausville EA, Weinstein JN, Friend S, Fornace Jr AJ, Kohn KW 1997 Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents Cancer Res 57:4285–4300
  35. Distler JH, Hagen C, Hirth A, Muller-Ladner U, Lorenz HM, del Rosso A, Michel BA, Gay RE, Nanagara R, Nishioka K, Matucci-Cerinic M, Kalden JR, Gay S, Distler O 2004 Bucillamine induces the synthesis of vascular endothelial growth factor dose-dependently in systemic sclerosis fibroblasts via nuclear factor-{kappa}B and simian virus 40 promoter factor 1 pathways. Mol Pharmacol 65:389–399[Abstract/Free Full Text]
  36. Pollmann C, Huang X, Mall J, Bech-Otschir D, Naumann M, Dubiel W 2001 The constitutive photomorphogenesis 9 signalosome directs vascular endothelial growth factor production in tumor cells. Cancer Res 61:8416–8421[Abstract/Free Full Text]
  37. Poulaki V, Mitsiades CS, McMullan C, Sykoutri D, Fanourakis G, Kotoula V, Tseleni-Balafouta S, Koutras DA, Mitsiades N 2003 Regulation of vascular endothelial growth factor expression by insulin-like growth factor I in thyroid carcinomas. J Clin Endocrinol Metab 88:5392–5398[Abstract/Free Full Text]
  38. Sartorius CA, Groshong SD, Miller LA, Powell RL, Tung L, Takimoto GS, Horwitz KB 1994 New T47D breast cancer cell lines for the independent study of progesterone B- and A-receptors: only antiprogestin-occupied B-receptors are switched to transcriptional agonists by cAMP. Cancer Res 54:3868–3877[Abstract]
  39. Lange CA 2004 Making sense of cross-talk between steroid hormone receptors and intracellular signaling pathways: who will have the last word? Mol Endocrinol 18:269–278[Abstract/Free Full Text]
  40. Leonhardt SA, Boonyaratanakornkit V, Edwards DP 2003 Progesterone receptor transcription and non-transcription signaling mechanisms. Steroids 68:761–770[CrossRef][Medline]
  41. Qiu M, Lange CA 2003 MAP kinases couple multiple functions of human progesterone receptors: degradation, transcriptional synergy, and nuclear association. J Steroid Biochem Mol Biol 85:147–157[CrossRef][Medline]
  42. Labriola L, Salatino M, Proietti CJ, Pecci A, Coso OA, Kornblihtt AR, Charreau EH, Elizalde PV 2003 Heregulin induces transcriptional activation of the progesterone receptor by a mechanism that requires functional ErbB-2 and mitogen-activated protein kinase activation in breast cancer cells. Mol Cell Biol 23:1095–1111[Abstract/Free Full Text]
  43. Meyer ME, Pornon A, Ji JW, Bocquel MT, Chambon P, Gronemeyer H 1990 Agonistic and antagonistic activities of RU486 on the functions of the human progesterone receptor. EMBO J 9:3923–3932[Abstract]
  44. Sartorius CA, Tung L, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone receptors bound to DNA are functionally switched to transcriptional agonists by cAMP. J Biol Chem 268:9262–9266[Abstract/Free Full Text]
  45. Liu Z, Auboeuf D, Wong J, Chen JD, Tsai SY, Tsai MJ, O’Malley BW 2002 Coactivator/corepressor ratios modulate PR-mediated transcription by the selective receptor modulator RU486. Proc Natl Acad Sci USA 99:7940–7944[Abstract/Free Full Text]
  46. Nilsen J, Brinton RD 2003 Divergent impact of progesterone and medroxyprogesterone acetate (Provera) on nuclear mitogen-activated protein kinase signaling. Proc Natl Acad Sci USA 100:10506–10511[Abstract/Free Full Text]
  47. Stoner M, Wormke M, Saville B, Samudio I, Qin C, Abdelrahim M, Safe S 2004 Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor {alpha} and SP proteins. Oncogene 23:1052–1063[CrossRef][Medline]
  48. Brenneisen P, Blaudschun R, Gille J, Schneider L, Hinrichs R, Wlaschek M, Eming S, Scharffetter-Kochanek K 2003 Essential role of an activator protein-2 (AP-2)/specificity protein 1 (Sp1) cluster in the UVB-mediated induction of the human vascular endothelial growth factor in HaCaT keratinocytes. Biochem J 369:341–349[CrossRef][Medline]
  49. Ryuto M, Ono M, Izumi H, Yoshida S, Weich HA, Kohno K, Kuwano M 1996 Induction of vascular endothelial growth factor by tumor necrosis factor {alpha} in human glioma cells. Possible roles of SP-1. J Biol Chem 271:28220–28228[Abstract/Free Full Text]
  50. Mueller MD, Vigne JL, Pritts EA, Chao V, Dreher E, Taylor RN 2003 Progestins activate vascular endothelial growth factor gene transcription in endometrial adenocarcinoma cells. Fertil Steril 79:386–392[CrossRef][Medline]
  51. Tang M, Mazella J, Gao J, Tseng L 2002 Progesterone receptor activates its promoter activity in human endometrial stromal cells. Mol Cell Endocrinol 92:45–53[CrossRef]
  52. Bar J, Lahav J, Hod M, Ben Rafael Z, Weinberger I, Brosens J 2000 Regulation of platelet aggregation and adenosine triphosphate release in vitro by 17ß-estradiol and medroxyprogesterone acetate in postmenopausal women. Thromb Haemost 84:695–700[Medline]
  53. Thomas T, Rhodin J, Clark L, Garces A 2003 Progestins initiate adverse events of menopausal estrogen therapy. Climacteric 6:293–301[Medline]
  54. Franke HR, Vermes I 2003 Differential effects of progestogens on breast cancer cell lines. Maturitas 46(Suppl 1):S55–S58
  55. Kalkhoven E, Kwakkenbos-Isbrucker L, de Laat SW, Van der Saag PT, van der BB 1994 Synthetic progestins induce proliferation of breast tumor cell lines via the progesterone or estrogen receptor. Mol Cell Endocrinol 102:45–52[CrossRef][Medline]
  56. Graham JD, Yeates C, Balleine RL, Harvey SS, Milliken JS, Bilous AM, Clarke CL 1996 Progesterone receptor A and B protein expression in human breast cancer. J Steroid Biochem Mol Biol 56:93–98[CrossRef][Medline]
  57. Ariga N, Suzuki T, Moriya T, Kimura M, Inoue T, Ohuchi N, Sasano H 2001 Progesterone receptor A and B isoforms in the human breast and its disorders. Jpn J Cancer Res 92:302–308[Medline]
  58. Bamberger AM, Milde-Langosch K, Schulte HM, Loning T 2000 Progesterone receptor isoforms, PR-B and PR-A, in breast cancer: correlations with clinicopathologic tumor parameters and expression of AP-1 factors. Horm Res 54:32–37[CrossRef][Medline]
  59. Sartorius CA, Shen T, Horwitz KB 2003 Progesterone receptors A and B differentially affect the growth of estrogen-dependent human breast tumor xenografts. Breast Cancer Res Treat 79:287–299[CrossRef][Medline]
  60. Richer JK, Jacobsen BM, Manning NG, Abel MG, Wolf DM, Horwitz KB 2002 Differential gene regulation by the two progesterone receptor isoforms in human breast cancer cells. J Biol Chem 277:5209–5218[Abstract/Free Full Text]
  61. Shyamala G, Yang X, Silberstein G, Barcellos-Hoff MH, Dale E 1998 Transgenic mice carrying an imbalance in the native ratio of A to B forms of progesterone receptor exhibit developmental abnormalities in mammary glands. Proc Natl Acad Sci USA 95:696–701[Abstract/Free Full Text]
  62. Conneely OM, Jericevic BM, Lydon JP 2003 Progesterone receptors in mammary gland development and tumorigenesis. J Mammary Gland Biol Neoplasia 8:205–214[CrossRef][Medline]
  63. Miller MM, James RA, Richer JK, Gordon DF, Wood WM, Horwitz KB 1997 Progesterone regulated expression of flavin-containing monooxygenase 5 by the B-isoform of progesterone receptors: implications for tamoxifen carcinogenicity. J Clin Endocrinol Metab 82:2956–2961[Abstract/Free Full Text]
  64. Lanari C, Molinolo AA 2002 Progesterone receptors—animal models and cell signalling in breast cancer. Diverse activation pathways for the progesterone receptor: possible implications for breast biology and cancer. Breast Cancer Res 4:240–243[CrossRef][Medline]
  65. Kordon EC, Molinolo AA, Pasqualini CD, Charreau EH, Pazos P, Dran G, Lanari C 1993 Progesterone induction of mammary carcinomas in BALB/c female mice. Correlation between progestin dependence and morphology. Breast Cancer Res Treat 28:29–39[CrossRef][Medline]
  66. Cappelletti V, Miodini P, Fioravanti L, Di Fronzo G 1995 Effect of progestin treatment on estradiol-and growth factor-stimulated breast cancer cell lines. Anticancer Res 15:2551–2555[Medline]
  67. Garcia T, Tuohimaa P, Mester J, Buchou T, Renoir JM, Baulieu EE 1983 Protein kinase activity of purified components of the chicken oviduct progesterone receptor. Biochem Biophys Res Commun 113:960–966[CrossRef][Medline]
  68. Migliaccio A, Piccolo D, Castoria G, Didomenico M, Bilancio A, Lombardi M, Gong WR, Beato M, Auricchio F 1998 Activation of the src/p21(ras)/erk pathway by progesterone receptor via cross-talk with estrogen receptor. EMBO J 17:2008–2018[Free Full Text]
  69. Pazdrak K, Olszewska-Pazdrak B, Liu T, Takizawa R, Brasier AR, Garofalo RP, Casola A 2002 MAPK activation is involved in posttranscriptional regulation of RSV-induced RANTES gene expression. Am J Physiol Lung Cell Mol Physiol 283:L364–L372
  70. Matthews JS, O’Neill LA 1999 Distinct roles for p42/p44 and p38 mitogen-activated protein kinases in the induction of IL-2 by IL-1. Cytokine 11:643–655[CrossRef][Medline]
  71. Fennessey PV, Pike AW, Gonzalez-Aller C, Horwitz KB 1986 Progesterone metabolism in T47Dco human breast cancer cells—I. 5{alpha}-Pregnan-3ß,6{alpha}-diol-20-one is the secreted product. J Steroid Biochem 25:641–648[CrossRef][Medline]
  72. Horwitz KB, Pike AW, Gonzalez-Aller C, Fennessey PV 1986 Progesterone metabolism in T47Dco human breast cancer cells—II. Intracellular metabolic path of progesterone and synthetic progestins. J Steroid Biochem 25:911–916[CrossRef][Medline]
  73. Wiebe JP, Lewis MJ 2003 Activity and expression of progesterone metabolizing 5{alpha}-reductase, 20{alpha}-hydroxysteroid oxidoreductase and 3{alpha}(ß)-hydroxysteroid oxidoreductases in tumorigenic (MCF-7, MDA-MB-231, T-47D) and nontumorigenic (MCF-10A) human breast cancer cells. BMC Cancer 3:9[CrossRef][Medline]
  74. Zhang L, Yu D, Hu M, Xiong S, Lang A, Ellis LM, Pollock RE 2000 Wild-type p53 suppresses angiogenesis in human leiomyosarcoma and synovial sarcoma by transcriptional suppression of vascular endothelial growth factor expression. Cancer Res 60:3655–3661[Abstract/Free Full Text]
  75. Shi Q, Le X, Abbruzzese JL, Peng Z, Qian C-N, Tang H, Xiong Q, Wang B, Li X-C, Xie K 2001 Constitutive SP-1 activity is essential for differential constitutive expression of vascular endothelial growth factor in human pancreatic adenocarcinoma. Cancer Res 61:4143–4154[Abstract/Free Full Text]
  76. Finkenzeller G, Sparacio A, Technau A, Marme D, Siemeister G 1997 SP1 recognition sites in the proximal promoter of the human vascular endothelial growth factor are essential for platelet-derived growth factor-induced gene expression. Oncogene 15:669–676[CrossRef][Medline]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
19/2/312    most recent
Author Manuscript (PDF)
Purchase Article
View Shopping Cart
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Request Copyright Permission
Google Scholar
Articles by Wu, J.
Articles by Hyder, S. M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Wu, J.
Articles by Hyder, S. M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals