University of Colorado Health Sciences Center Department of Medicine Division of Endocrinology Denver, Colorado 80262
INTRODUCTION
The effects of progesterone in the breast are controversial due to the structural and developmental complexity of this organ. Depending on the experimental model system, the cell context, and the duration of treatment, progesterone can elicit either proliferative or antiproliferative effects on breast epithelial cell growth. The multiple secondary hormonal factors, which regulate breast cell growth and development in combination with progesterone, contribute to this conundrum. We propose that these seemingly contradictory effects of progesterone can be explained by virtue of its ability to act as a priming factor for the actions of secondary agents. The purpose of this minireview is to highlight recent data suggesting that the strength and duration of progesterone signaling determine whether it acts as a proliferative or inhibitory agent and that progestin pretreatment sets the stage for enhanced activity of locally acting cytokines and growth factors through synergy with their signaling pathways. Our intention is not to review the extensive literature regarding the growth-altering properties of progesterone; we refer the reader to several reviews on the subject (1, 2, 3, 4). Rather, here we would like to develop the concept that progesterone is a priming factor in the context of breast cancer progression; progesterone pretreatment promotes a switch from growth driven primarily by steroid hormones to growth driven primarily by peptide growth factors. Thus, the priming effects of progesterone may contribute, in part, to the development of steroid hormone resistance during breast cancer progression. It is our hope that these ideas will invite further discussion regarding the role of progesterone in breast cancer growth and progression, thus complementing the much better understood role of estradiol in this process.
PROGESTERONE AND BREAST CELL PROLIFERATION
The requirement for progesterone in normal mammary gland lobular-alveolar development is well established (5). However, its role in the mature, precancerous, and cancerous breast remains poorly defined. During the cyclical hormonal changes that characterize the normal menstrual cycle, breast epithelium undergoes increased DNA synthesis associated with mitosis. This peaks in the late luteal phase, at a time when circulating levels of progesterone are highest, providing evidence that progesterone is a proliferative hormone in the breast. Consistent with this observation, the high progesterone levels during pregnancy induce further lobular-alveolar development in preparation for lactation, indicating both a proliferative and differentiative function for this hormone. Other studies of the effects of progesterone on normal breast epithelial cell growth have, however, produced equivocal results (reviewed in Refs. 2, 4). Progesterone increases DNA synthesis in organ culture, decreases or has no effect on the growth of explants in nude mice, and decreases proliferation in primary cell cultures of normal breast epithelium and cultured breast cancer cells (reviewed in Ref. 1). In vivo studies involving treatment of patients with high doses of progesterone before breast surgery show fewer mitotic figures when compared with estrogen alone, or estrogen plus progesterone, and high-dose progestins are effective second-line therapies for patients whose tumors are hormone responsive. Apparently, progesterone can be both proliferative and antiproliferative. Is there a unifying hypothesis that can reconcile this paradox?
PROGESTERONE EFFECTS ON THE CELL CYCLE: ROLE OF TREATMENT TIME AND DOSE
Studies using human breast cancer cell lines have provided
valuable insight into the paradoxical effects of progestins on cell
proliferation, with the demonstration of clear biphasic effects on cell
cycle progression (4, 6, 7). Hissom and Moore (8) first reported the
proliferative effect of progesterone in T47D cells. Indeed, the
immediate response of asynchronous cultured T47D human breast cancer
cells to a single pulse of progesterone is proliferative (Fig. 1); there is transient induction of genes
associated with cell cycle progression, with acceleration of cells
through one mitotic cycle. Early (012 h) changes include increased
hyperphosphorylated pRb, increased expression of cyclins D1, D3, E, A,
and B, activation of cyclin-CDK2 and -CDK4 complexes, and induction of
c-myc and c-fos mRNAs (9). Levels of the
cyclin-dependent kinase (CDK) inhibitors, p21 and p27, gradually rise
during the proliferative phase of progestin treatment, peaking after
the end of the first G1/S transition (6). Interestingly,
induction of cyclin D1 alone, using an inducible promoter expression
system, mimics the effects of progestins and accelerates
G1-phase progression with kinetics similar to those of
progesterone (10). Studies using transgenic and knockout mice have
further demonstrated the absolute requirement for cyclin D1 activation
in the mitogenic response of the mammary gland to progestins (11, 12).
Progestins have also been reported to decrease expression of the p53
tumor suppressor protein, possibly contributing further to their
proliferative effects (13). In sum, a single pulse of progesterone is
transiently growth stimulatory.
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Based on this biphasic response to a pulse of progesterone, we
propose that transient or intermittent doses of progesterone are growth
stimulatory, while continuous or sustained high-dose progesterone is
growth inhibitory (Fig. 1). Such a model has far-reaching implications
for the timing of progestin treatments in clinical settings and
predicts that the effects of continuously administered progestins
differ significantly from those of episodically or cyclically
administered progestins. It may also explain why physiological levels
of progesterone may have different proliferative effects than high-dose
progestins. This model also suggests that the endogenous cyclical
progesterone of the menstrual cycle may have different physiological
consequences than the continuous progesterone of pregnancy. Clearly
then, the dose and timing of progestin treatments may prove to be
critical in determining the experimental or clinical outcome.
PROGESTERONE PRIMES CELLS FOR THE ACTIONS OF GROWTH FACTORS
The sequential stimulatory, followed by inhibitory, effects of
progestins on breast cancer cell growth suggest that the biphasic
response is part of a single signaling cascade. Sutherland et
al. (4) suggest that one round of cell division followed by acute
growth inhibition at the G1/S boundary reflects
progestin-induced differentiation, a one-process model. However, they
also recognize the possibility that the biphasic effects of progestins
on breast cell growth are mediated by distinct mechanisms that, under
differing conditions, lead to predominance of either a stimulatory or
inhibitory response pathway. Recent results from our laboratory appear
to blend these two possibilities (6, 15, 16). We propose that
progesterone acts as a priming agent. In this scenario, progesterone
treatment brings about a dual response: first, it drives cells to a
decision point at the G1/S boundary, and second, it induces
cellular changes that permit other factors, possibly tissue-specific,
to influence the ultimate proliferative or differentiative state of the
cells. Whether to grow, differentiate, or die (as in the case of
involution at the end of lactation), is thus determined by the cell
context and the endogenous hormonal milieu. This model suggests that
breast cells can be directed toward one path or another by cross-talk
between various signaling pathways and the progesterone/PR complex
(Fig. 1). We outline below some evidence for such cross-talk.
A clear demonstration of the concept that progesterone can
function as a priming factor comes from studies involving pretreatment
of breast cells with progesterone, followed by treatment with growth
factors or cytokines. For example, despite the fact that epidermal
growth factor (EGF) receptors are present and functional in T47D human
breast cancer cells, EGF is not mitogenic in progestin-naive cells.
However, after priming by progestins for approximately 48 h, the
cells become highly sensitive to the proliferative effects of EGF,
despite loss of pRb protein and high levels of p21 (6). This response
is perhaps not surprising, since progestin-mediated up-regulation of
EGF receptors and other type I tyrosine kinase receptor family
members (c-erbB2 and c-erbB3) is well documented; overexpression of
these receptors is predictive of a poor prognosis in breast cancer
patients (reviewed in Ref. 17). Progestins also up-regulate PRL
receptors, insulin-like growth factor receptors, transforming
growth factor- receptors, fibroblast growth factors, and insulin
receptors (reviewed in Ref. 17), thereby perhaps pushing cells toward a
proliferative path in the presence of the appropriate
growth-stimulatory ligands, which can bypass the G1/S block
produced by progesterone alone (Fig. 1
). For example, while
progesterone alone inhibits the growth of T47D cells, cotreatment with
insulin leads to synergistic induction of cell growth (18, 19).
Estrogen and progesterone augment growth responsiveness of mammary
tissue to cAMP, via increases in cAMP-dependent protein kinase activity
(20). Thus, progesterone can greatly increase the sensitivity of breast
cells to locally acting mitogens. Furthermore, in addition to
increasing the sensitivity of cells to growth factors by increasing the
levels of their receptors, progestins also enhance the sensitivity of
their downstream signaling pathways (see below).
CONVERGENCE OF PROGESTERONE SIGNALING WITH GROWTH FACTOR AND CYTOKINE SIGNALING
Progestin/PR-mediated sensitization of breast cancer cells to growth factors and cytokines occurs at multiple levels in mitogenic signaling cascades and contributes to a biochemical switch in growth regulation (15, 16). We will discuss two distinct pathways through which progesterone/PR amplify epidermal growth factor and cytokine signaling: one involves mitogen- activated protein kinase (MAPK), the other involves signal transducers and activators of transcription (STATs).
MAPKs
How does EGF push progestin-arrested cells past the
G1/S boundary in the face of low hypophosphorylated pRb and
rising p21/p27 levels? We recently reported that pretreatment with
progestins can selectively potentiate EGF-stimulated MAP kinase
activities, leading to synergistic up-regulation of cell cycle proteins
required for transition past the G1/S boundary (Fig. 2A). Interestingly, EGF has no effect on
cyclin E expression in progestin-naive cells, but up-regulates cyclin E
after progestin pretreatment. Cyclin E is required for entry into
S-phase and, in contrast to cyclin D1, cyclin E can promote
G1/S transition even in cells lacking functional pRb
protein (21). Accumulation of active cyclin E/CDK2 complexes is
controlled by Myc and Ras (22). Progestins are known to increase the
expression of c-myc (4, 9), and the human c-myc promoter
contains a progesterone response element (PRE) (4, 23). This may
explain how EGF, via Ras/MAPKK/MAPK activation, can regulate
cyclin E only in progestin-pretreated cells (Fig. 2
), where
c-myc is also up-regulated. Similarly, only
progesterone-pretreated T47D breast cancer cells undergo proliferation
in response to EGF, despite low levels of hypophosphorylated pRb (6).
We speculate that EGF-stimulated cell-cycle reentry of cells that are
growth arrested after progestin treatment may be mediated by a MAPK-
and cyclin E-dependent, pRb-independent, pathway. Consistent with this
idea is the finding that inhibition of MAPK by the MEK inhibitor
(PD98059) in the presence of progestin and EGF results in levels of
p21, cyclin D1, and cyclin E that fall below the control levels seen
with EGF alone (Fig. 2B
). Thus, while the levels of cyclin D1, cyclin
E, and p21 are up-regulated by progestins alone in a MAPK-independent
manner, pretreatment with progestin followed by EGF synergistically
up-regulates these proteins via a MAP kinase-dependent mechanism. These
findings support the concept that, as a consequence of progestin
pretreatment or priming, the regulation of key cell cycle proteins
switches from MAPK-independent to MAPK-dependent mechanisms.
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Consistent with the concept of progesterone priming, Stat5 proteins and mRNAs are up-regulated during pregnancy, when progesterone levels are high (discussed in Ref. 16). Indeed, EGF and PRL can only induce tyrosine phosphorylation of Stat5 in T47D breast cancer cells after progestin pretreatment (16). Like PR and cyclin D1, Stat5a is required for complete mammary gland lobular alveolar growth and lactation (26). Other transcription factors, such as members of the CCAAT/enhancer-binding protein family (C/EBP) are also up-regulated by progesterone in a PR-dependent manner (J. K. Richer and K. B. Horwitz, in preparation). C/EBP-ß is involved in lobular alveolar outgrowth during differentiation of the mammary gland (27, 28). The remarkable temporal importance of PR, cyclin D1, Stat5, and C/EBP-ß in normal growth and development of the mammary gland suggests the intriguing possibility that disregulation of these factors contributes to breast pathology.
Of note, the progestin-induced changes in growth factor/cytokine receptors, and STAT and C/EBP levels, occur after induction of cell-cycle proteins and the transient proliferative burst caused by a single dose of progesterone (see above). These changes are therefore unlikely to be involved in the early growth response. Rather, progesterone may sensitize normal or malignant breast cells to subsequent actions of growth factors and/or cytokines, with the potential for synergistic responses. Thus, in addition to cross-talk at the level of cell-surface receptors, and at the level of their signaling intermediates, this model suggests a third level of regulation directly on the transcription complex.
SYNERGISTIC ACTIVATION OF GROWTH-REGULATORY GENES BY PROGESTERONE AND GROWTH FACTORS
Although growth factors/cytokines function at the cell surface and
steroid hormones act primarily in the nucleus, triggering different
signal transduction pathways, the resulting signals often converge on
the same subset of genes. Transcriptional synergy on the ß-casein
promoter in the presence of PRL and glucocorticoids is mediated via an
interaction between glucocorticoid receptors (GR) and Stat5 (29).
Transcriptional synergy between EGF and progesterone has been reported
using a minimal promoter containing a PRE as well as on the complex
PREs of the mouse mammary tumor virus (MMTV) promoter (30). We recently
reported transcriptional synergy between EGF and progesterone on the
promoters that drive the growth-regulatory genes, c-fos and
p21 (Fig. 3 and Ref. 16), neither of
which contains a PRE. However, both STAT and C/EBP sites are present in
these promoters. When transfected into breast cancer cells, the
c-fos and p21 promoters are modestly responsive to
progesterone or EGF alone, while both hormones added simultaneously
elicit a profound synergistic response (Fig. 3
). We showed that the
progesterone response of the p21 promoter is mediated by tethering of
PR to transcription factor Sp1 and CBP (31). Similarly, glucocorticoid
regulation of p21 (32, 33) and other promoters (34) lacking
glucocorticoid response elements maps to C/EBPß sites. Thus, evidence
is accumulating that PR and GR can regulate the activities of genes
that are simultaneously regulated by growth factors, even when the
promoters lack classical DNA binding sites for the steroid receptors.
The ability of GR and the AP-1 and NF
B transcription factors to
interact on target promoters lacking direct binding sites for at least
one of the factors has recently been reviewed (35). Transcriptional
cross-talk on promoters that lack DNA- binding sites for steroid
receptors may be a more common mechanism of steroid hormone action than
previously thought. Of note is the additional observation that
cross-talk does not always imply transcriptional synergy; frequently,
cross-talk results in interference between the actions of two hormonal
agents, as in the case of glucocorticoid repression of the inflammatory
response mediated by interleukins and interferons (reviewed in Ref.
35). Clearly, further studies are required to define the consequences
of such complex interactions.
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The role of p21 in the dual control of breast cancer cell growth
by progestins and EGF is of interest. In T47D cells, increased p21
expression is associated with cell growth arrest in response to
progestins alone (discussed above). However, after release from this
arrest by EGF, p21 levels increase even further (Fig. 2), and
progesterone plus EGF synergize to enhance p21 promoter-driven
transcription (Fig. 3
). This effect of progesterone plus EGF appears to
be paradoxical, given the classical CDK-inhibitory/differentiative
properties of p21. It is now recognized, however, that p21 has
multifunctional actions that can contribute to antidifferentiative,
antiapoptotic, or proliferative cellular responses. For example, forced
expression of p21 inhibits terminal differentiation of primary mouse
keratinocyctes, a function that is separable from the ability of p21 to
inhibit cyclin-CDK complexes (36). Furthermore, p21 binds to and
inactivates JNK (37), a mediator of apoptosis in certain systems;
elevated levels of p21 may contribute to androgen-independent growth of
prostatic carcinoma cells via an antiapoptotic mechanism (38).
Additional proliferative actions of p21 may be explained by the finding
that at low concentrations, p21 acts as a nucleation factor that
assembles and activates cyclin D-CDK kinase complexes, while higher
concentrations of p21 inhibit a constant level of cyclin-associated CDK
activity (39). Since progestin plus EGF treatment up-regulates
G1/S-phase cyclins, concomitantly up-regulated p21 may
participate in cyclin-CDK activation (39). Thus, in breast cancer
cells, multiple complex functions of p21 may contribute to restarting
the cell cycle machinery in response to secondary growth signals
initiated by EGF or cytokines, after progestin pretreatment and cell
cycle arrest.
SUMMARY
In the breast, data from numerous laboratories
suggest that cross-talk exists between PR and growth factor and
cytokine signaling pathways at multiple levels (Fig. 4). At the cell surface (level 1),
progestins up-regulate growth factor and cytokine receptors. We have
expanded this observation by examining the effects of progestins in the
cytoplasm (level 2) where progestins regulate several intracellular
effectors by increasing the levels and altering the subcellular
compartmentalization of Stat5, increasing the association of Stat5 with
phosphotyrosine-containing proteins and tyrosine phosphorylation of
JAK2, Cbl, and Shc, and potentiating EGF-stimulated p42/p44 MAPKs, p38
MAP kinase, and JNK activities. Together, these events lead to
sensitization of downstream signaling pathways to the actions of
locally acting secondary factors. Finally, inside the nucleus (level
3), agonist-occupied PR synergize with nuclear transcription factors
that are growth-factor regulated, to control the activity of key genes
involved in breast cell fate (Figs. 1
and 4
). We speculate that after
progesterone treatment, orchestrated combinations of steroid hormones
and growth factors or cytokines can fine tune the timing and degree of
expression of a subset of genes that determine whether progestin-primed
cells undergo proliferation, differentiation, or programmed cell
death.
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ACKNOWLEDGMENTS
We gratefully acknowledge J. Dinny Graham, Ph.D. (University of Colorado Health Sciences Center, Department of Medicine, Division of Endocrinology) and Margaret C. Neville, Ph.D. (University of Colorado Health Sciences Center, Department of Physiology and Biophysics) for helpful comments on this manuscript.
FOOTNOTES
Address requests for reprints to: Carol A. Lange, University of Colorado Health Sciences Center, Department of Medicine, Division of Endocrinology, Campus Box B151, 4200 East Ninth Avenue, Denver, Colorado 80262. E-mail: Carol.Lange{at}UCHSC.edu
This work was supported by NIH Grants DK-53825 (to C.A.L.), CA-26869 (to K.B.H.), and DK-48238 (to K.B.H.).
Received for publication February 11, 1999. Revision received March 10, 1999. Accepted for publication March 12, 1999.
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