Progesterone-Independent Effects of Human Progesterone Receptors (PRs) in Estrogen Receptor-Positive Breast Cancer: PR Isoform-Specific Gene Regulation and Tumor Biology
Britta M. Jacobsen,
Stephanie A. Schittone,
Jennifer K. Richer and
Kathryn B. Horwitz
Departments of Medicine, Pathology and the Molecular Biology Program, University of Colorado Health Sciences Center, Aurora, Colorado 80045
Address all correspondence and requests for reprints to: Britta M. Jacobsen, Ph.D., Department of Medicine/Endocrinology, University of Colorado School of Medicine, Mail Stop 8106, PO Box 6511, Aurora, Colorado 80045. E-mail: Britta.Jacobsen{at}uchsc.edu.
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ABSTRACT
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Progesterone receptors (PRs) are prognostic markers in breast cancers irrespective of the patients progestational status. However, there are two PR isoforms, PR-A and PR-B, that are equimolar in the normal breast but dysregulated in advanced disease. Postmenopausal, tamoxifen-treated patients with estrogen receptor (ER)-positive, PR-A-rich tumors have much faster disease recurrence than patients with PR-B-rich tumors. To study the mechanisms we engineered ER+ breast cancer cells that express each PR isoform under control of an inducible promoter. We identified 79 genes regulated by progesterone (P), mainly by PR-B, and 51 genes regulated without progesterone, mainly by PR-A. Only nine genes were regulated with and without ligand, leading to definition of three classes: I) genes regulated only by liganded PR; II) genes regulated only by unliganded PR; III) genes regulated by both. Unliganded PR-A and PR-B differentially regulate genes that coordinate extracellular signaling pathways and influence tumor cell biology. Indeed, in the absence of P, compared with ER+/PR-B+ or PR cells, ER+, PR-A+ cells exhibit an aggressive phenotype, are more adhesive to an extracellular matrix, and are more migratory. Additionally, unliganded PR-A and PR-B both inhibit cell growth and provoke resistance to Taxol-induced apoptosis. We propose that PR-A:PR-B ratios, even in the absence of P, influence the biology and treatment response of ER+ tumors, that PR-A isoforms are functionally dominant in P-deficient states, and that PR-A rich tumors are especially aggressive.
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INTRODUCTION
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PRESENCE OF PROGESTERONE receptors (PRs) in estrogen receptor (ER)-positive breast cancers is associated with good prognosis (1) and indicates that tumors are highly likely to respond to estrogen (E) suppression therapies targeted at ER (e.g. tamoxifen or aromatase inhibitors) (2). However, whereas ER+/PR+ tumors are likely to be hormone responsive, ER+/PR tumors tend to be unresponsive (3). Why should the response to therapies targeted at ER in ER+ tumors be influenced by presence or absence of PR? Also of interest is the fact that ER/PR+ tumors are more aggressive, and patients have a poorer outcome, than patients with ER+/PR+, ER+/PR, or ER/PR tumors (4, 5). The reasons for this are unknown but suggest that PRs have a more complex role in breast cancer than was previously recognized, i.e. that PRs are more than simply markers of ER function (6).
These issues are complicated further by the fact that normal and malignant tissues contain two PR isoforms, PR-B and N-terminally truncated PR-A, that differ extensively in their ability to regulate transcription in the presence of progesterone (P), with PR-B much stronger (7). Total breast PR levels are regulated not only by Es, but also by mixed antiestrogens like tamoxifen (8, 9, 10, 11, 12). Regardless of levels, the breasts of normal women express equimolar ratios of PR-A:PR-B (13). However, not only the levels, but also PR-A:PR-B ratios are altered by exogenous E and E+P used in hormone replacement therapies, or by tamoxifen used in cancer treatments (12). Additionally, whereas PR-A:PR-B are equimolar in the normal breast (13), the ratio is increasingly dysregulated as breast cancers progress so that more than 70% of metastatic tumors have an excess of PR-A or PR-B (13, 14). The significance of this is still under study, but PR-A excess has been associated both with poor clinical outcome (13) and with more rapid disease recurrence after tamoxifen treatment (14). In this regard, it may also be important that the normal breasts of women with germ-line mutations of the breast cancer susceptibility genes BRCA1 and BRCA2 exhibit an abnormal abundance of PR-A (15). Thus, PR-A excess and cancer susceptibility are intricately linked, and it appears that PR-A excess is also harmful once cancer has developed. Interestingly, most of the deleterious effects of PR-A in cancers are observed in P-deficient, postmenopausal women.
Several studies assessing the role of the two PRs in the presence of P use experimental mouse models. However, unlike the equimolarity of humans, normal mouse mammary glands have a 3:1 excess of PR-A over PR-B (16). Nevertheless, knockout models have been useful. They demonstrate that PRs are absolutely required for normal mammary gland development (17), and that PR-B alone allows relatively normal although prematurely arrested development (18) whereas PR-A alone leads to premalignant developmental changes (19). Similarly, transgenic mice that overexpress PR-A exhibit mammary gland hyperplasia and disorganized basement membranes characteristic of early malignancy (20, 21). These studies describing differential effects of PR-A vs. PR-B use intact mice with, presumably, physiological levels of P.
On the other hand, clinical studies reporting differential effects of PR-A vs. PR-B usually involve P-deficient women (14). Similarly, studies of human tumor xenografts grown in ovariectomized, athymic mice show that PR-A and PR-B differentially influence E-dependent ER+ tumor growth in the absence of P (22). This led us to propose that some of the differential effects of the two PRs occur in a ligand-independent (LI) manner. There is precedence for this. An interesting recent study suggests that the ancestral steroid receptor functioned in a LI manner (23). Several receptors including ERs, PRs, and androgen receptors (ARs) can be activated by cross-talk with cell surface-signaling pathways, a step that bypasses their hormones (24). Transcription by unliganded PR, mostly nonhuman, can be activated by signaling pathways including cAMP, phorbol esters, dopamine, epidermal growth factor (EGF), and phosphatase inhibitors (24, 25, 26, 27) and is inhibitable by RU486 (28). Mechanisms for convergence of growth factor and cytokine signaling pathways with PR have focused on phosphorylation. For example, EGF activates PR in the absence of P via MAPK phosphorylation of Ser294 in the PR N terminus (29). It is also likely that altered phosphorylation of PR coregulators is involved in LI activation (30).
Because clinical and experimental studies point to LI functions for PR, and to compare PR-A vs. PR-B signaling in the absence of P, we engineered ER+ human breast cancer cells in which each PR isoform is under control of an inducible promoter (28). These models permit analysis of gene and biological effects of each PR in the identical cell background, before and after receptors have been induced, under conditions in which exogenous hormone levels can be tightly controlled. In this paper we: 1) demonstrate that PRs regulate transcript levels in the absence of P; 2) show that PR-A isoforms are the dominant LI receptors; 3) define gene classes regulated by unliganded PRs; and 4) show effects of unliganded PRs on the biology of ER+ breast cancer cells.
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RESULTS
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Transcript Regulation by PR in the Absence (LI) and Presence (LD) of P
PRs Regulate mRNA Levels in a LI and LD Manner.
The present study uses three new (28) ER+, PR cell lines in which PR-A, PR-B, or an empty expression vector are under inducible control of the ecdysone analog, ponasterone A (ponA). The cells are called Y iA (PR-A+), Y iB (PR-B+), and Y iNull (PR). The immunoblot (Fig. 1A
) shows that all three cell lines are PR in the absence of inducer. In Y iA and Y iB, 24 h of ponA induces PR-A or PR-B to levels similar to those seen in wild-type T47Dco cells, which is equivalent to the average of levels in breast cancers (14). Induced PRs bind ligand with high affinity (not shown). The Y iNull cells remain PR during ponA treatment. All three cell lines are ER
positive (Fig. 1B
).

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Fig. 1. Inducible T47D Cell Lines Express PR-A or PR-B upon Treatment with ponA
A, Y iNull, Y iA, and Y iB cells were treated with vehicle () or 10 nM ponA (+) for 24 h. These cells as well as wild-type T47Dco were harvested and lysed, and whole cell extracts were resolved by SDS-PAGE and immunoblotted with anti-PR antibodies. B, Aliquots of the same extracts were resolved on SDS-PAGE and probed with anti-ER or anti-PSTAIR antibodies.
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The three cell lines were profiled for endogenous transcript expression patterns in either the PR or PR+ state, and in the absence or presence of 6 h P. Thus, four conditions were tested per cell line: 1) PR-, P-; 2) PR, P+; 3) PR+, P; and 4) PR+, P+. Each condition was assessed in all three cell lines in triplicate, time-separated experiments. Chips interrogating approximately 10,000 human gene transcripts were analyzed, and mRNAs that were regulated at least 1.5-fold, three of three times in a statistically significant manner, were scored. The dendrograms in Fig. 2
are an overview of the results. Figure 2A
shows gene clusters regulated in the three cell lines when they are in either the PR or PR+ state, in the absence of P. Importantly, cells expressing unliganded PR-A or PR-B segregate into distinct branches from cells that lack PR, suggesting that expression of unliganded PR alters the phenotype of cells sufficiently to distinguish them from PR cells. As is evident from the striking increase in red, unliganded PR-A isoforms regulate more genes than unliganded PR-B isoforms.

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Fig. 2. Dendrograms Showing LD and LI PR Regulation of Transcript Expression Levels
A, LI genes. Y iNull, Y iA, and Y iB cells growing in defined medium with twice charcoal-stripped serum were treated 24 h with DMSO () or ponA (+) to generate PR-negative or PR-positive cells. None of the cells received P (). B, LD genes. Y iNull, Y iA, and Y iB cells growing in defined medium with twice charcoal-stripped serum, were treated 24 h with ponA to induce no PR, PR-A, or PR-B, respectively, and then treated 6 h with ethanol () or 10 nM P (+). In both sets, cellular RNA was prepared and HU95 Affymetrix gene chips interrogating approximately 10,000 unique human genes were probed as described. GeneSpring 5.0 and 6.0 were used to classify genes expressed in the 12 experimental conditions, each analyzed in triplicate, time-separated sets into distinct statistically significant subclasses. Branches on the left designate relatedness of each cell type. Cell types were clustered based on distance. Each column represents a single gene, and cell type and treatments are specified for each row. Red signifies above average gene expression, green indicates below average expression, and black signifies average gene expression.
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The dendrogram in Fig. 2B
shows the effects of P (6 h) in PR+ cells. P treatment segregates PR+ cells into branches of the dendrogram different from cells expressing unliganded PR or no PR (Y iNull controls). In the presence of hormone, more gene transcripts are regulated by PR-B than PR-A. This is opposite to the results with unliganded PR.
Summary of PR mRNA Regulation Patterns.
Several subgroups were identified by their regulation patterns, four of which are illustrated in Fig. 3
: 1) transcripts up-regulated only by liganded PR (class I); 2) transcripts up-regulated only by unliganded PR (class II); 3) transcripts up-regulated by unliganded PR and further up-regulated by P (class IIIa); or 4) down-regulated by P (class IIIb). Other patterns parallel these four but involve initial down-regulation (data not shown). Expression patterns of several genes in each class were confirmed by RT-PCR. One example of each class of regulation is shown in Fig. 3
both by its expression array pattern (main panel), as well as by its RT-PCR confirmation (inset under each panel). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control for the PCR data. The four specific genes shown are TNF receptor-associated factor 5 (TRAF5) (class I), OGG1 (class II), DR6 (class IIIa), and SNK (class IIIb). Also indicated in Fig. 3
are the total number of genes in each class, and the distribution of their regulation among PR-A only, PR-B only, or both PRs. All the genes are tabulated and discussed in Tables 1
and 2
. Each class is discussed further below.

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Fig. 3. Expression Profiles of Four Human Genes, Representing Four Classes of PR Regulation
Transcripts shown are: class I, TRAF5 regulated by PR-B; class II, OGG1 regulated by PR-A; class IIIa, TNFRSF21 regulated by PR-A; class IIIb; SNK regulated by PR-B. Array profling: The graphs show normalized expression levels for each mRNA, in the absence (no PR) and presence of PR (PR+), and the absence and presence (+) of P, as calculated using GeneSpring 5.0. Red lines denote the LI component of each expression profile. Vertical bars represent SE. RT-PCR (inset): For each of the four mRNAs, RT-PCR was used to confirm its regulation pattern in the four PR and treatment states, and GAPDH was used as an internal control. Also indicated are the number of genes in each class and their PR isoform regulatory profile. The number of genes in this figure is somewhat higher than the number in Fig. 4 because several genes fall into two classes depending on how each PR isoform regulates them and are therefore counted twice here. Five genes (TNFRSF21, CDKN1A, MAP2K3, ELL2, and NDRG1) are in two classes and are therefore classified according to their regulation by each isoform. For example, NDRG1, which is regulated by PR-A in a class IIIa pattern and by PR-B in a class I pattern, is counted twice in this figure but not included among the eight class I genes regulated by both PRs.
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Class I: Ligand-Dependent (LD) Genes.
Class I gene transcripts (Table 1
) are regulated by PR in a classical manner, i.e. only in the presence of P. Of the approximately 10,000 genes probed, 73 were defined as P regulated in a class I manner: 56 (77%) uniquely by PR-B, nine (12%) uniquely by PR-A, and only eight (11%) by both, with the majority of these (six of eight) more strongly by PR-B. Clearly, liganded PR-B isoforms regulate more gene transcripts than PR-A isoforms and the minimal overlap between the two PRs indicates important functional differences between them. A 1.5-fold cut-off was chosen for these highly reproducible, statistically significant, results. Table 1
lists the LD genes, the class into which they fall, and the extent of their transcript regulation by PR-A and/or PR-B. The dominance of PR-B regulation is evident. Many of the LD genes regulated by inducible PR were also regulated in cells stably expressing PR (Table 1
footnotes and Ref. 7). One example is TRAF5, a TNF receptor-associated factor. Its LD regulation was confirmed by RT-PCR (Fig. 3
). Three genes, well regulated in a LI manner, are only 1.2- to 1.3-fold further regulated by ligand (class IIIa) but are included in Table 1
because the LD effect was highly reproducible and statistically significant.
Class II: LI Genes.
The transcripts of class II genes are regulated only by unliganded PRs, with P having no further effect (Table 2
). Among 45 LI genes regulated in a class II manner, 26 (58%) are regulated only by PR-A, three (7%) are regulated only by PR-B, and 16 (36%) are regulated by both, with the majority of the latter (11 of 16) regulated more strongly by PR-A. This is the opposite of the class I pattern. Expression of two genes was down-regulated by unliganded PR; both genes by PR-A. Among the up-regulated genes is OGG1, an N-glycosylase/AP-lyase involved in the repair of oxidative DNA base damage. Regulation of several LI genes was confirmed by RT-PCR in at least three independently derived clones of inducible cells. Regulation of OGG1 by unliganded PR-A is shown in Fig. 3
. Table 2
lists genes the transcripts of which are regulated by unliganded PR. It also defines the class each belongs to and the PR-A vs. PR-B distribution, demonstrating the dominant role of PR-A. The table is color-coded. Red and pink predominance illustrates extensive LI regulation of effects emanating from the cell membrane: ones targeted to the outside involved in cell-cell signaling, extracellular matrix (ECM) and cell-cell adhesion, or motility and invasiveness; and ones targeted intracellularly like signaling molecules. Many metabolic genes are also regulated in a LI-independent manner, as are some genes involved in cell growth and apoptosis.
Class III: Mixed Genes.
Transcripts of class IIIa genes (Fig. 3
and Table 2
) are up-regulated by LI PR, and further up-regulated by P. Four genes were in this category, all regulated by PR-A. One is DR6 (TNFRSF21), a death domain containing orphan receptor member of the TNF receptor superfamily, the regulation of which by unliganded and liganded PR-A was confirmed by RT-PCR (Fig. 3
). Class IIIb genes are up-regulated by LI PR and down-regulated by P. Four such genes were found, one regulated by PR-A and three by PR-B. An example is the serum-inducible kinase SNK, regulated by PR-B and confirmed by RT-PCR (Fig. 3
). Finally, a few genes belong to more than one class. For example, NDRG1 is a class I gene when regulated by PR-B and a class IIIa gene when regulated by PR-A.
Venn diagrams (Fig. 4
) summarize gene up-regulation patterns by PR, with each gene counted once. Among approximately 10,000 unique transcripts probed, 121 or approximately 1.2%, are PR up-regulated. They fall into LD and LI subgroups with little (only nine of 121) overlap between them. This strongly indicates that genes regulated in a LI manner represent an entirely different subset, having different functional consequences than LD ones. The LD genes are regulated mainly by PR-B; the LI genes are regulated mainly by PR-A, suggesting that PR-A isoforms are functionally dominant in P-deficient states. Because unliganded PRs regulate multiple genes involved in adhesion, motility, growth, and migration, we asked whether they influence these aspects of breast cancer cell biology.

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Fig. 4. Summary of P-Dependent vs. -Independent PR-Regulated Genes, Showing the Extent of Overlap among Sets
Expression profiles for approximately 10,000 genes were screened as described, and Venn diagrams were generated in GeneSpring (versions 5.0 and 6.0). Among 121 PR-regulated genes only nine are regulated by both LD and LI mechanisms. The majority of P-regulated genes involve PR-B; the majority of LI genes involve PR-A.
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Unliganded PR and Tumor Cell Biology
Subcellular Localization of Unliganded PR.
Figure 5
demonstrates that at low and intermediate protein expression levels, both PR-A (Fig. 5A
) and PR-B (Fig. 5B
) are exclusively nuclear in the absence of P. Within nuclei, unliganded PRs localize to punctate foci that may be active sites of transcription (31, 32). Interestingly, DNA-binding deficient PRs also localize to nuclei but do not patch at foci (data not shown). We also find that despite clonal origin of the cells, there is variability in PR levels after ponA induction, with 2030% remaining PR and the rest expressing low, intermediate, or high PR levels. Figure 5C
shows a cell overexpressing PR-A, which displays both nuclear and cytoplasmic staining. Although some studies (33) indicate that only PR-B isoforms localize to the cytoplasm, in our experience, both PR-A and PR-B spill into the cytoplasm, but only if they are overexpressed.

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Fig. 5. After Their Synthesis, Unliganded PR-A and PR-B Concentrate in Nuclei Unless Receptors Are Overexpressed
Y iA (panels A and C) and Y iB (panel B) cells growing on cover slips in twice charcoal-stripped serum-containing medium were treated with vehicle (DMSO) or 10 µM ponA for 24 h to induce PR. Cells were incubated with a primary anti-PR antibody (DAKO 1294) that recognizes both PR-A and PR-B, followed by a secondary green fluorescent fluorescein isothiocyanate (FITC) antibody, and cell nuclei were stained with blue fluorescent 4'6-diamidino-2-phenylindole (DAPI). Cells were photographed at x100 magnification. Representative deconvolved Z stack images are shown.
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Unliganded PR-A Isoforms Alter Cell Morphology.
Figure 6A
shows two independent fields (set 1 and set 2) assessing morphology of cells grown without P, when in the PR state and after 8 d of PR-A or PR-B induction. PR and PR-B+ cells appear healthy and rounded, but PR-A+ cells exhibit greatly increased numbers of cellular processes and branching. Quantitation of an equivalent cell number is shown in Fig. 6B
. Compared with Y iB cells in the uninduced and PR-B+ state, which exhibit little branched morphology, PR-A induction in Y iA cells increases processes and branches almost 3-fold. This change, classified as aggressive (34, 35), was observed in some cells within 48 h of PR-A acquisition.

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Fig. 6. Breast Cancer Cells Expressing Unliganded PR-A Have an Aggressive Morphology
A, Uninduced (no PR) or ponA-induced Y iA cells (PR-A+), and ponA-induced Y iB cells (PR-B+) were grown 8 d in the absence of P. Multiple-phase contrast microscopy fields were photographed. Two fields are shown for each condition. B, Seventeen different fields for uninduced or induced Y iA ( 425 cells per condition) and 10 fields for Y iB ( 210 cells) were counted for branching and process formation. The asterisk denotes significance to P < 0.05 compared with no PR.
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Unliganded PRs Influence Cell Growth, Adhesion, and Migration.
Many genes regulated by unliganded PR, including NDRG1 and p21 (CDKN1A), control proliferation (36). We observe a small, but statistically significant (P < 0.05), decrease in cell growth after PR-A or PR-B induction compared with similarly treated Y iNull cells (data not shown). Several genes encoding cell adhesion molecules, including CD44, DSCAM, PCDH1, IGFBP5, and ITGA3, are up-regulated by unliganded PR-A. Figure 7
shows that on BSA, basal adhesion levels of all three cell lines is unaltered by PR induction. ECM components, Matrigel and Collagen, increase basal adhesion in all cells in the absence of PR. However, when PRs are switched on, only PR-A increase adhesiveness to ECM further, another property associated with aggressiveness (37, 38). Transcript expression of two genes that regulate cell motility, ARHC and MYL9, are up-regulated by unliganded PR-A. Figure 8
shows the migratory behavior of the three cell lines when they are PR. Compared with basal levels, migration increases significantly (asterisk) only when PR-A isoforms are expressed.

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Fig. 7. PR-A-Containing Cells Adhere More Strongly to an ECM than PR-B-Containing or PR-Negative Cells
Y iA, Y iB, or Y iNull cells were induced with DMSO (no PR) or ponA (PR-A or PR-B) for 24 h. Cells were then incubated 2 h in 96-well plates coated with BSA, Matrigel (MG), or collagen (col), and attached cells were fixed, stained with crystal violet, and counted using a microplate reader. Statistical analysis was performed using ANOVA followed by Tukeys post test; *, P < 0.05.
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Fig. 8. Unliganded PR-A Isoforms Increase the Motility of Breast Cancer Cells
Y iA, Y iB, or Y iNull cells were induced with DMSO (no PR) or ponA (PR-A or PR-B) for 48 h. Equal numbers (6250) of cells per condition were plated in duplicate on top of 8-µm pore Transwells that were coated on the underside with collagen. After fixation and staining, the number of cells that migrated to the collagen in 22 h was quantified. The average number of cells per filter is shown. *, P < 0.05 using Students t test. Experiments were performed three times, and data from a representative experiment are shown.
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Unliganded PRs Promote Taxol Resistance.
Transcripts of several genes regulated by unliganded PR (DR6, CDKN1A, TGFBI, CDC6, and NDRG1) have been implicated in growth and apoptosis (36). To assess effects of unliganded PR on apoptosis, we tested Taxol, a potent apoptosis-inducing chemotherapeutic agent used to treat breast cancers (39). The three cell lines were treated without or with Taxol, in either the PR or PR+ state (Fig. 9
). In the absence of PR ("no") and Taxol (), each cell exhibits different basal apoptosis levels, with Y iA relatively low, and Y iB and Y iNull higher. Taxol alone (+) strongly increased apoptosis in all three PR cells. Induction of PR alone did not alter apoptosis rates. However, the strong Taxol-induced apoptosis seen in PR cells was attenuated by presence of PR. Thus, unliganded PRs render cells resistant to Taxol-induced apoptosis. That PR cells are more sensitive to apoptosis than PR+ cells was confirmed by poly (ADP-ribose) polymerase cleavage and annexin V staining (data not shown). In the clinic, Taxol is reportedly more effective in patients whose tumors are ER/PR (39, 40, 41). Our models allow detailed study of this important clinical issue.

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Fig. 9. Unliganded PR-A or PR-B Attenuate Taxol-Induced Apoptosis
Y iA, Y iB, or Y iNull cells were induced with DMSO (no PR) or ponA (PR-A or PR-B) for 48 h, and then treated 48 h with RPMI or RPMI + Taxol as shown. To measure apoptosis, a nucleosomal fragmentation assay was performed as described in Materials and Methods. Each experiment was performed three times for each cell type, and a representative result is shown. *, P < 0.05 as determined by ANOVA followed by Tukeys post test.
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DISCUSSION
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We previously described construction and properties of the PR-inducible cells (28). They are ideal models because they serve as their own controls, with the null cells providing an extra layer of control. Additionally, because PRs are usually E-regulated proteins, the cells circumvent the confounding effects of E by using ponA to regulate PR. This is important because E and P often have opposing actions, as, for example, on cell migration (Fig. 8
) (42). The PR levels achieved by an appropriate time and dose of ponA (Fig. 1
) are the average of PRs in breast cancers (14) with some cell-cell variability. Expression of select gene transcripts is similar at low and high PR levels (data not shown), but effects of varying PR levels on cell biology require further study.
Because the induction system uses a highly modified Drosophila ecdysone receptor (VgRXR) not found in mammalian cells, binding sites should not exist in mammalian promoters. Indeed, neither LD PR transcriptional regulation of PRE2-luciferase nor the ligand dose required for maximal activity is affected by VgRXR activation (data not shown). Nevertheless, array profiling identified three endogenous genes regulated by VgRXR, and eight regulated by VgRXR plus PR (data not shown). Mechanisms for such regulation could include the rare convergence of random DNA sequences that bind VgRXR directly or that tether VgRXR through other bound factors. This underscores the need for tight controls when using array profiling. Here we demonstrate that not only in the presence of P, but also in the absence of exogenous P, PRs regulate transcript expression levels of a subset of genes and influence breast cancer cell biology.
LD mRNA Regulation
Many of the genes we identified as LD in cells stably expressing PR-A or PR-B (7) are also LD in cells with inducible PRs. However, the correlation is not perfect. This could have simple explanations including use of different gene chips, changes in probe sets and probe set sequences, and major changes in software and statistical algorithms (43). More interesting explanations could be invoked, however. It has been reported that a stably integrated promoter cannot be activated by transiently expressed liganded PR but is well regulated by stably expressed PR (44). The same may be true for some endogenous genes. Their activation may require mature PRs that have accumulated posttranslational modifications such as phosphorylation (45, 46, 47) or sumoylation (48), which occur many hours after initial synthesis. Inducible PRs allow us to study such questions.
LI mRNA Regulation
Although PRs are classically defined as "ligand-activated transcription factors" they also influence transcript expression levels in the absence of P. Transcription is inhibitable by the antiprogestin RU486, suggesting direct receptor effects (28). This is likely to be physiologically important in P-deficient states, i.e. after menopause, with E replacement therapy, prepuberty, in men, in cancer, in association with drug addiction, etc. (49, 50, 51). LI regulation was presumed to be impossible because of receptor occupancy by heat shock proteins (52), but unliganded heat shock protein-free PRs localize in nuclei (53, 54), including to foci (Fig. 6
) that may be active sites of transcription.
In addition to PR, other steroid receptors, including ERs and ARs, are activated in a LI manner (55). Unliganded ARs bind DNA and activate transcription, which is enhanced by coexpression of steroid receptor coactivator 1, receptor-associated coactivator 3, and p300 (56). Transcription by unliganded ER involves phosphorylation of three N-terminal serine residues (57), allowing the receptors to interact with coactivators like the DNA repair enzyme, MMS19 (58). Effects of unliganded PRs were first observed in the rodent brain after receptor activation by dopamine (26) and later by phosphatase inhibitors (24). We have shown that EGF activates PR via MAPK phosphorylation of Ser294 in the N terminus (29, 59, 60). In fact, unliganded steroid receptors are activated in a variety of tissues by many factors including IL-6 (61), dopamine (26), heregulin ß1 (62), cAMP, and EGF (25). All are believed to activate kinase cascades that modify receptor or coactivator phosphorylation states (30). Differential phosphorylation of LI and LD PR could account, in part, for their functional differences (45).
Unliganded PR-A vs. PR-B
Studies to date addressing the role of PR on mammary gland development, cell morphology, adhesion, and migration have of necessity used P. The mouse PR knockout and transgenic models all assess effects of PR isoforms in the presence of P (18, 19, 20), as do cell culture models that describe differential effects of PR isoforms on tumor biology (63). Here we document regulation by unliganded PR of genes encoding proteins involved in ECM binding, cell-cell communication, and membrane signaling, including small ion transporters important in homeostasis. Examples of genes regulated by unliganded PR-A and PR-B include major vault protein associated with increased tumor size and nodal metastases (64, 65), Stanniocalcin 1, a marker of breast cancer progression (66), and TGFBI and COL6A1, encoding cell aggressiveness markers (67). However, most genes are regulated only by unliganded PR-A including ones encoding ECM adhesion molecules such as DSCAM, ITGA3, CD44, PCDH1, and IGF-binding protein 5 (IGFBP5) and cell aggressiveness markers such as ITGA3 (67).
ECM adhesion and aggressiveness may be related. For example, IGFBP5 promotes tight junction formation and apoptosis at the onset of normal breast involution but promotes ECM adhesion while protecting cells from apoptosis in the malignant breast (68). IGFBP5 is one of 70 breast cancer genes, the increase of which correlates with metastasis and poor prognosis (69). Also up-regulated only by unliganded PR-A is RhoC (ARHC), the expression of which is linked to breast tumor invasiveness associated with small tumor size (70). Thus, the propensity for growth vs. metastasis may be unrelated. Inflammatory breast cancers, highly aggressive forms of locally advanced disease, overexpress RhoC GTPase in more than 90% of cases (71). RhoC has also been classified as a mammary oncogene conferring a metastatic phenotype (72).
Unliganded PR and Chemotherapy
Pre- and postmenopausal women with ER/PR breast tumors respond well to chemotherapies, whereas ER+/PR+ tumors tend to be resistant (73). Efficacy of these treatments is closely associated with apoptosis (74). Several genes involved in apoptosis are regulated by unliganded PR. They include DR6 (TNFRSF21), a member of the death domain-containing receptor family, the overexpression of which is observed in prostate and breast cancers (75). It is induced by unliganded PR-A, and by liganded PR-A and PR-B. It promotes apoptosis in ER/PR HeLa cervicocarcinoma cells but not in ER+/PR+ MCF-7 breast cancer cells (76). Another death receptor, DR5 (TNFRSF10B), is regulated by liganded PR-A. The DNA repair enzyme OGG1 (77), up-regulated by unliganded PR-A and PR-B, may provide additional resistance to apoptosis and DNA damage. Thus, our data suggest several mechanisms for further study that underlie the Taxol resistance of PR+ cells (Fig. 9
).
Unliganded PR, Breast Cancer Growth, and Metastasis
There are considerable data suggesting that unliganded PRs influence breast cancers. Even in P-deficient states, ER+/PR+ cancers are much more likely to respond to E suppression therapies, such as antiestrogens or aromatase inhibitors, than ER+/PR cancers (3). If the treatments target ER, why should the absence of PR make a difference? Until recently, it had been assumed that unliganded PRs are simply passive markers of ER activity, lacking independent function. Newer studies indicate, however, that PRs are independent markers of disease prognosis irrespective of progestational state (1, 78). We have shown that growth of ER+ breast cancer cells into E-dependent tumors in ovariectomized nude mice is inhibited by PR despite P deficiency (22).
It is also believed, based on experimental models, that ER+/PR+ breast cancers are well differentiated, presenting as low-risk, well-defined lesions. However, ER+/PR+ tumors can, and do, metastasize (79, 80, 81). The myoepithelial phenotype of PR-A+ cell (Fig. 6A
) suggests that they are likely to be invasive, poorly differentiated, and aggressive (34, 35, 82). Unliganded PR-A isoforms also increase cell adhesion to ECM (Fig. 7
), believed to prime cells for stromal invasion, and metastasis (37, 38). We therefore speculate that PR-A-rich tumors or ones that contain only PR-A (14) are especially aggressive. This has important clinical implications because PR-A excess is common in advanced disease (83), and tamoxifen-treated patients with PR-A-rich tumors demonstrate more rapid disease recurrence due either to heightened aggressiveness or accelerated hormone resistance (14).
We propose that in ER+ states, unliganded PRs influence breast cancer cell biology. If so, class II genes (Fig. 3
and Table 2
) may be useful to screen tumors for functional PR, especially in postmenopausal women. Additionally, expression profiling could be used to identify not only PR+ tumors but possibly to define their PR isoform ratios based on isoform-specific gene expression. In sum, our data indicate that each PR isoform is functionally different in the presence and absence of hormone and underscore the complexity of the actions of P, at least in breast cancers.
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MATERIALS AND METHODS
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Cell Culture
Expression of PR-A (Y iA cells), PR-B (Y iB cells), or an empty vector (Y iNull cells) is controlled by a modified Drosophila ecdysone receptor (VgRXR) activated by the synthetic ecdysone analog, ponA, in ER+, PR T47D human breast cancer cells (28). Cells are routinely cultured in MEM containing 7% fetal bovine serum plus other additions (28). For PR induction, cells are harvested, replated in MEM containing 7% twice charcoal-stripped serum, and treated 24 h with vehicle [dimethylsulfoxide (DMSO)] or 10 µM ponA.
Gene Expression Profiling
Poly A+ RNA was prepared from total RNA and expression profiling performed using HG-U95Av2 (Affymetrix, Santa Clara, CA) arrays (28). Experiments were done in triplicate for each of three cell lines and four treatment groups, using time-separated samples. Data analyses used Microarray Suite (Affymetrix, version 5.0) and GeneSpring software (versions 4.2.1, 5.0, and 6.0; Silicon Genetics, San Carlos, CA). LI genes were defined by comparing PR cells treated 24 h with DMSO vs. ponA. LD genes were defined by comparing ponA-induced, PR+ cells treated 6 h with ethanol vs. 10 nM P. At 6 h of P, approximately 55% of gene transcripts are directly PR regulated as shown by cycloheximide treatment (7). Statistical analyses used Students t test and/or one-way ANOVA followed by a Tukey post test (P < 0.05). Fold changes for statistically significant genes were calculated from the average of raw expression data generated by Microarray Suite. Dendrograms were generated in GeneSpring using hierarchical clustering, and similarity was measured using a distance correlation. Microarray data are published as supplemental data on The Endocrine Societys Journals Online web site at http://mend.endojournals.org.
RT-PCR
Regulation of several transcripts was confirmed by RT-PCR. cDNA was synthesized from total RNA of independent experimental sets (28). Primer sequences were: SNK forward (fwd): 5'-CTAAGGCATACAGTTCTTGACTTTGGACA-3', SNK reverse (rev): 5'-GAATGCACTTTTCCAGCCACAAGTA-3', hOGG1 fwd: 5'-GAGCTGCGCCTGGACCTGGTTCTGCC-3', hOGG1 rev: 5'-GAATTTCTGAGCCACCTCTTGGAAG-3'; TRAF5 fwd: 5'-GACTTTGAGCCCAGTATAGA-3'; TRAF5 rev: 5'-CCCAGAATAACCTTGGCATT-3'; DR6 fwd: 5'-GGGCTTCTTCGTGGATGAGTCGGAGC-3'; DR6 rev: 5'-CCCGCAGCTCCTCAGGATTTAG-3'. GAPDH was run in parallel as an internal control as described previously (7).
Immunoblotting
Whole-cell extracts (200 µg) were resolved on a 7.5% denaturing polyacrylamide gel, transferred to nitrocellulose, blocked, probed for PR with a mixture of AB-52 and B-30 antibodies (84), for ER with Ab-15 (Neomarkers, Fremont, CA), or for PSTAIR using a rabbit polyclonal antibody Anti-cdk1/cdc2 (Upstate Biotechnology, Inc., Lake Placid, NY), and visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL).
Immunocytochemistry and Confocal Microscopy
Cells (60,000) were plated on coverslips, grown in MEM plus twice-dextran-coated charcoal-stripped serum, induced or not with ponA, washed with PBS, fixed in MeOH/acetone at 25 C, rewashed, and placed in 10% normal goat serum (NGS) in PBS for 1 h. After rewashing, mouse antihuman PR primary antibody (PgR 1294, DAKO Corp., Carpinteria, CA) was added at a 1:100 dilution in 1% NGS overnight at 4 C. Coverslips were washed and incubated 2.5 h at 25 C in secondary Alexa Fluor 488 goat antimouse antibody (Molecular Probes, Eugene, OR) diluted 1:100 in 1% NGS. Cells were counterstained with 4'6-diamidino-2-phenylindole and mounted on slides using Fluoromount G. Fluorescent images were captured using an Olympus IX70 inverted microscope (Olympus Corp., Lake Success, NY) and a Photometrics Quantix camera (Photometrics Ltd., Tucson, AZ) at x100 magnification. Images were deconvolved using a Silicon Graphics O2 computer with DeltaVision software, and one representative Z stack image is shown.
ECM Adhesion Assay
Cells growing in MEM plus twice charcoal-stripped serum were induced 24 h with ponA, and then harvested and resuspended at 5 x 105 cells/ml. Plates (96 well) were coated with Matrigel (3 µg/well) or collagen (20 µg/well) for 1 h at 37C and then washed with PBS followed by 1% BSA for 30 min at 37 C. Wells were rinsed and 5 x 104 cells per well were plated in triplicate for 2 h at 37 C. Wells were washed, and adherent cells were fixed with 100 µl 1% glutaraldehyde (5 min at room temp), stained with 0.1% crystal violet, washed, and lysed with 1% sodium dodecyl sulfate. Plates were read at 570 nm on an MRX microplate reader (Dynatech Laboratories, Billinghurst, W. Sussex, UK), and absorbance values were averaged. Statistical significance was determined using one-way ANOVA followed by Tukeys post test (P < 0.05). Assays were performed at least three times, and one representative experiment is shown.
Apoptosis
Cells (5000/well) were plated in 96-well plates. PRs were induced (ponA) or not (DMSO) for 48 h at least three times. The medium was then replaced with serum-free RPMI without or with 0.02 µg/ml Taxol for 48 h (Paclitaxel; Bristol-Myers Squibb, Princeton, NJ), and apoptosis was quantified (Cell Death Detection ELISAPLUS; Roche, Indianapolis, IN). Results are representative of three independent experiments.
Migration
Cells were treated with DMSO or 10 µM ponA for 48 h, harvested, pelleted, and resuspended in serum-free RPMI. Cells (6250/well) in duplicate per treatment group were added to 8-µm pore Transwells (Corning/Costar, Corning NY) coated on the underside with collagen (5 µg/ml). RPMI containing 0.5% twice charcoal-stripped serum was placed in the bottom chamber as a chemoattractant. Migration proceeded for 22 h. Cells were fixed, stained with Hema3 (Fisher Scientific, Pittsburgh, PA), and four separate fields on two independent filters per treatment group were photographed and quantified using Image Pro (MediaCybernetics, Silver Spring, MD). Assays were performed at least three times and one representative experiment is shown.
Cell Morphology
Cells (60,000) were plated onto coverslips in MEM, treated with DMSO or ponA for 48 h, and then switched to phenol red-free/serum-free media for 8 d with continuous DMSO or ponA. Seventeen (Y iA) or 10 (Y iB) fields per treatment group were photographed and quantified with Image Pro from several independent experiments. The average number of nuclei per field for Y iA and Y iB cells was 25 and 21, respectively. The average number of processes + branches per field normalized to the total number of nuclei is shown. Statistical significance was calculated using one-way ANOVA followed by Tukeys post test (*, P < 0.05 compared with the same cell in the PR-uninduced state).
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ACKNOWLEDGMENTS
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We thank Dr. Carla Van Den Berg for reagents and help with apoptosis assays, Drs. Lynne Bemis and Melissa Allen for advice on migration assays, Steven Fadul for confocal microscopy advice, Ted Shade for preliminary data analysis, and the University of Colorado Cancer Center Gene Expression and Light Microscopy Core Facilities. Special thanks to Robert W. Burke and Lynne A. Griffin for thoughtful input and advice.
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FOOTNOTES
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This work was supported by National Institutes of Health Grant CA26869, Department of Defense Research Service Command Grant BC981225, National Foundation for Cancer Research Grant 10COL3, the Avon Products Foundation, and National Research Service Award postdoctoral fellowship F32 CA90073 (to B.M.J.).
First Published Online November 24, 2004
Abbreviations: AR, androgen receptor; DMSO, dimethylsulfoxide; E, estrogen; ECM, extracellular matrix; EGF, epidermal growth factor; ER, estrogen matrix; fwd, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGFBP5, IGF-binding protein 5; LD, ligand dependent; LI, ligand independent; NGS, normal goat serum; P, progesterone; ponA, ponasterone A; PR, progesterone receptor; PR-A, progesterone receptor A isoform; PR-B, progesterone receptor B isoform; rev, reverse; TRAF5, TNF receptor-associated factor 5.
Received for publication July 15, 2004.
Accepted for publication November 15, 2004.
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