Estrogen and Antiestrogen Regulation of Cell Cycle Progression in Breast Cancer Cells

Endocrine-Related Cancer (2003) 10 179–186

International Congress on Hormonal Steroids and Hormones and Cancer Estrogen and antiestrogen regulation of cell cycle progression in breast cancer cells

S F Doisneau-Sixou1,2, C M Sergio1, J S Carroll 1, R Hui 1, E A Musgrove1 and R L Sutherland 1

1Cancer Research Program, Garvan Institute ofMedical Research, St Vincent’s Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia 2De´partement ‘Innovation The´rapeutique et Oncologie Mole´culaire’, Centre de Physiopathologie de Toulouse Purpan, INSERM U563 and Institut Claudius Regaud, 20–24 rue du Pont St Pierre, 31052 Toulouse cedex, France (Requests for offprints should be addressed to R L Sutherland; Email: [email protected])

Abstract The central involvement ofestrogen in the development ofthe mammary gland and in the genesis ofbreast cancer has lent impetus to studies ofthe links between estrogen action and the cell cycle machinery. Recent studies ofthe estrogenic regulation ofmolecules with known roles in the control of

G1/S phase progression have resulted in signiﬁcant advances in understanding these links. Estrogens independently regulate the expression and functionofc-Myc and cyclin D1 and the induction ofeither c-Myc or cyclin D1 is sufﬁcient to recapitulate the effects of estrogen on cell cycle progression. These pathways converge at the activation ofcyclin E–Cdk2 complexes. The active cyclin E–Cdk2 complexes are depleted ofthe cyclin dependent kinase (CDK) inhibitor p21 WAF1/CIP1 because of estrogen-mediated inhibition ofnascent p21 WAF1/CIP1. Insulin and estrogen synergistically stimulate cell cycle progression, and the ability ofestrogen to antagonize an insulin-induced increase in p21WAF1/CIP1 gene expression appears to underlie this effect. Antiestrogen treatment of MCF-7 cells leads to an acute decrease ofc-Myc expression, a subsequent decline in cyclin D1, and ultimately arrest ofcells in a state with featurescharacteristic ofquiescence. An antisense-mediated decrease in c-Myc expression results in decreased cyclin D1 expression and inhibition ofDNA synthesis, mimicking the effects of antiestrogen treatment and emphasizing the importance of c-Myc as an estrogen/antiestrogen target. These data identify c-Myc, cyclin D1, p21WAF1/CIP1 and cyclin E-Cdk2 as central components ofestrogen regulation ofcell cycle progression and hence as potential downstream targets that contribute to the role ofestrogen in oncogenesis. Endocrine-Related Cancer (2003) 10 179–186

Introduction late transcription through ‘non-classical’ response sites that bind heterologous transcription factors including Sp1, c-Jun/ Sex steroid hormones have a major role in the growth and ATF-2 and AP-1, probably via protein–protein interactions development of target tissues including the mammary gland since ERs do not bind these sites directly (McDonnell & where they interact with other hormones, growth factors and Norris 2002). Increasing evidence indicates that E2 induces cytokines in the precise regulation of proliferation and differ- nongenomic effects (often immediate and transient) via sig- entiation. The classical model of 17β-estradiol (E2) action naling pathways more commonly associated with growth involves ligand-mediated activation of the nuclear estrogen factor activation of cell surface receptors (Cato et al. 2002). receptors ERα and ERβ, which interact directly with estro- For example, a nuclear receptor-interacting protein that gen response elements (ERE) in the promoters of target modulates ERα interaction with the Src family and conse- genes and recruit various coactivators to mediate transcrip- quently affects ER transcriptional activity has recently been tional regulation (McDonnell & Norris 2002). ERs also regu- identiﬁed (Wong et al. 2002).

Endocrine-Related Cancer (2003) 10 179–186 Online version via http://www.endocrinology.org 1351-0088/03/010–179  2003 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/28/2021 01:29:17AM via free access Doisneau-Sixou et al.: Estrogen and antiestrogen action in breast cancer

The cell cycle phase-specificity of the effects of estro- berg 1997, Prall et al. 1997). A particularly powerful model gens on proliferation and differentiation have focused atten- system has been breast cancer cells growth-arrested in the tion on the role of estrogens and their receptors in regulating G0/G1 phase by pretreatment with antiestrogens and then processes controlling the entry into, progression through, and stimulated by estrogen treatment to progress semi- exit from the G1 phase of the cell cycle. Central to these synchronously through the remainder of the G1 phase and cell cycle control mechanisms are cyclin D1-Cdk4 and cyclin into S phase (Osborne et al. 1984, Planas-Silva & Weinberg E-Cdk2, which phosphorylate substrates including the prod- 1997, Prall et al. 1997). The use of specific estrogen antagon- uct of the retinoblastoma susceptibility gene pRB, thereby ists to achieve cell synchrony in this experimental design per- allowing initiation of DNA synthesis (Weinberg 1995). mits selective magnification of ER-mediated effects relative They are controlled by several mechanisms including: tran- to more generalized responses to cell cycle progression, and scriptional activation of cyclin expression, regulatory phos- we have used this model system to dissect the molecular phorylation of the cyclin dependent kinase (CDK) subunit by events involved in estrogen stimulation of cell proliferation. kinases including the CDK-activating kinase (CAK) and the Cdc25 family of phosphatases, and interactions with mem- Regulation of c-Myc expression bers of two distinct families of CDK inhibitors of which p16INK4A and p21WAF1/CIP1 are prototypic (Sherr & Roberts The mitogenic, apoptotic and oncogenic functions of c-Myc 1999). Essentially all of these modes of regulation have been depend upon dimerization with the heterologous protein documented following estrogen treatment. Another well- Max, DNA binding and transcriptional regulation, suggesting studied target of estrogen action with a role in the control of that c-Myc acts by regulating genes involved in cell prolifer- cell cycle progression is c-Myc, a nuclear phosphoprotein of ation and/or apoptosis (reviewed in Oster et al. 2002). These the basic helix-loop-helix family of transcription factors. The functions require the C-terminal dimerization domain, but it recent expansion of knowledge on the molecular mechanisms has now become evident that the N-terminal region of c-Myc regulating rates of cell cycle progression has provided a also plays an essential role, particularly in the regulation of framework within which to develop deeper insight into the cell cycle progression and transformation. The N-terminal mechanistic basis of estrogen-induced mitogenesis and anti- region contains a transcription regulation domain, spanning estrogen action in breast cancer cells. This brief review sum- about 150 amino acids, with two evolutionarily conserved marizes recent developments in this area. motifs named Myc box I and Myc box II (Oster et al. 2002). In rat uteri (Murphy et al. 1987) and breast cancer cell lines (Dubik et al. 1987), the rapid and direct regulation of Experimental models c-myc by estrogen, apparently via an atypical ERE (Dubik & Estrogens and antiestrogens would be expected to exert Shiu 1992), places c-myc induction amongst the earliest opposite effects on the same targets given that antiestrogens detectable transcriptional responses to E2. Strong evidence are competitive inhibitors of the ER-mediated actions of that c-Myc is likely to play a key role in estrogen action is estrogens; thus a greater depth of understanding of the provided by the demonstration that c-Myc antisense oligo- molecular mechanisms that mediate the antiproliferative nucleotides inhibit estrogen-stimulated breast cancer cell pro- action of antiestrogens also yields insights into estrogen liferation (Watson et al. 1991) and that induction of c-Myc action. The growth inhibitory effects of antiestrogens in in antiestrogen-arrested cells can mimic the effects of estro- ERα-positive breast cancer cells are profound, and this gen by re-initiating cell cycle progression (Prall et al. 1998). allowed early demonstration of a G1 phase site of action for Further experiments with c-Myc mutants lacking Myc box I, antiestrogens (Osborne et al. 1983, Sutherland et al. 1983). Myc box II or the entire N-terminal domain indicate that the Studies using synchronized cells demonstrated that cells were N-terminal region of c-Myc including Myc box II is essential most sensitive to estrogens and antiestrogens in the early G1 for c-Myc-induced cell cycle progression in this model (CM phase, immediately following mitosis (Taylor et al. 1983, Sergio, EA Musgrove & RL Sutherland, unpublished data). Leung & Potter 1987, Musgrove et al. 1989), compatible with a model whereby estrogens and antiestrogens acting via Regulation of cyclin D1 expression and the ER regulate the rate of progression through the early G1 function phase of the cell cycle. The growth stimulatory effects of estrogens in cell cul- Treatment of breast cancer cells with antiestrogens inhibits ture have often been subtle and heavily dependent on the pRB phosphorylation (Watts et al. 1995) while estrogen experimental system employed, and thus several methods of induces significant increases in the phosphorylation of this cell synchronization have been employed to increase the pro- key substrate of cyclin–CDK complexes (Altucci et al. 1996, portion of cells in the estrogen-sensitive G1 phase of the cell Foster & Wimalasena 1996, Hurd et al. 1997, Planas-Silva & cycle (Osborne et al. 1984, Leung & Potter 1987, Altucci et Weinberg 1997, Prall et al. 1997). This indicates that the al. 1996, Foster & Wimalasena 1996, Planas-Silva & Wein- cyclin–CDK complexes active in G1 phase are likely to be

180 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/28/2021 01:29:17AM via free access Endocrine-Related Cancer (2003) 10 179–186 targets of estrogen action. In particular, cyclin D1, which increase in cyclin D1 mRNA (Altucci et al. 1996, Prall et al. binds to and activates both Cdk4 and Cdk6, has been impli- 1997), indicating that cyclin D1 is directly regulated by ER. cated in estrogen/antiestrogen regulation of cell cycle pro- However, the cyclin D1 promoter region does not contain a gression. Cyclin D1 expression declines rapidly following classical ERE. Instead, induction by ERα has been mapped exposure to growth-inhibitory antiestrogens (Musgrove et al. to a cAMP response element (CRE) close to the transcription

1993, Watts et al. 1995), while E2 treatment of MCF-7 breast start site (Sabbah et al. 1999, Castro-Rivera et al. 2001) and cancer cells that have been growth arrested by a variety of a more distal Sp1 site bound by both ERα and Sp1 in strategies is followed by pronounced increases in cyclin D1 chromatin immunoprecipitation assays (Castro-Rivera et al. protein expression, cyclin D1–Cdk4 association and Cdk4 2001). An AP-1 site has partly overlapping functions with activity (Altucci et al. 1996, Foster & Wimalasena 1996, the CREalthough it mediates a relatively weak response on Planas-Silva & Weinberg 1997, Prall et al. 1997). The its own (Liu et al. 2002). Interestingly, ERα and ERβ have increase in cyclin D1 expression occurs after the earliest opposing actions on the cyclin D1 promoter although both changes in c-Myc expression, but coincides with increased are transcriptional activators at ERE-containing promoters phosphorylation of pRB and precedes S phase entry by some (Liu et al. 2002). 9 h (see Fig. 1). Compelling evidence that cyclin D1 plays an essential The effect of estrogen on cyclin D1 protein expression role in estrogen-induced cell cycle progression comes from appears to be predominantly transcriptionally mediated, since studies in which cyclin D1 is either functionally inhibited, increased expression of cyclin D1 mRNA precedes changes or its expression is enforced ectopically. Thus, when either in cyclin D1 protein (Altucci et al. 1996, Prall et al. 1997). antibodies against cyclin D1, or the Cdk4-speciﬁc inhibitor Inhibitors of protein synthesis block the estrogen-induced p16INK4A are introduced into MCF-7 cells by microinjection,

Figure 1 A model of estrogen effects on molecules regulating G1 phase progression in MCF-7 cells. See text for details. D1, cyclin D1; E, cyclin E; p21, p21WAF1/CIP1. Shaded cyclin – CDK complexes are inactive. www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021181 01:29:17AM via free access Doisneau-Sixou et al.: Estrogen and antiestrogen action in breast cancer

estrogen fails to stimulate G1/S phase progression (Lukas et Separation of the cyclin E–Cdk2 complexes by gel fil- al. 1996), indicating that cyclin D1 is necessary for estrogen tration chromatography indicated that estrogen treatment was action. Conversely, induced expression of cyclin D1 in associated with the formation of high molecular weight com- MCF-7 or T-47D breast cancer cells that have been growth- plexes (Prall et al. 1997), and that induction of c-Myc or arrested in G1 phase by pretreatment with antiestrogens is cyclin D1 likewise led to the formation of active high followed by Cdk4 activation, increased phosphorylation of molecular weight cyclin E–Cdk2 complexes (Prall et al. pRB and subsequent S phase entry, thereby mimicking the 1998). These complexes constituted a minority of the cyclin actions of estrogen (Wilcken et al. 1997, Prall et al. 1998). E-Cdk2 protein but were of high specific activity, accounting for the majority of cyclin E-Cdk2 activity, and relatively Estrogen activation of cyclin E–Cdk2 deficient in p21WAF1/CIP1 and p27Kip1. Estrogen treatment WAF1/CIP1 holoenzyme complexes and CDK inhibitor relieves the inhibitory activity of p21 towards cyclin involvement E-Cdk2 (Planas-Silva & Weinberg 1997, Prall et al. 1997), the result of a decrease in newly synthesized p21WAF1/CIP1, In addition to activation of cyclin D1–Cdk4 complexes, which has a greater inhibitory activity than pre-existing estrogen also activates cyclin E–Cdk2 complexes within p21WAF1/CIP1 (Prall et al. 2001). The repression of p21WAF1/CIP1 ȁ3 h, substantially preceding entry into S phase (Foster & during estrogen-induced proliferation may involve transcrip- Wimalasena 1996, Planas-Silva & Weinberg 1997, Prall et tional repression by c-Myc since p21WAF1/CIP1 is a c-Myc al. 1997). This is in contrast to cell cycle progression stimu- target (Gartel et al. 2001, van de Wetering et al. 2002) and lated by other mitogens in which cyclin E-Cdk2 activation is is down-regulated after c-Myc induction in this system (CM associated with G1/S phase transition (Ho & Dowdy 2002), Sergio, EA Musgrove & RL Sutherland, unpublished suggesting that cyclin E-Cdk2 has a particularly important results). The pRB-related protein p130, which can compete role in estrogen-induced cell cycle progression. However, with p21WAF1/CIP1 for cyclin-CDK binding (Zhu et al. 1995, following estrogen treatment there is little or no change in Shiyanov et al. 1996) is recruited to the cyclin E–Cdk2 com- the levels of cyclin E, Cdk2, or the CDK inhibitors, plex following estrogen treatment and c-Myc or cyclin D1 p21WAF1/CIP1 and p27Kip1 in either total cell lysates or the cyclin induction, contributing to the increased size of the complex E–Cdk2 complexes prior to entry into S phase (Prall et al. and the decreased association with p21WAF1/CIP1 (Prall et al. 1997). The mechanistic basis for this effect is thus not readily 1998, see Fig. 1). apparent and has subsequently been explored in detail Supporting a role for Cdc25A in estrogen action, anti- (Planas-Silva & Weinberg 1997, Prall et al. 1997, 2001). sense Cdc25A oligonucleotides inhibited estrogen-induced Some mechanistic insights into the activation of cyclin Cdk2 activation and DNA synthesis while inactive cyclin E– E-Cdk2 by estrogen are provided by studies utilizing MCF-7 Cdk2 complexes from p16INK4A-expressing, estrogen-treated cells that contain either inducible c-Myc or cyclin D1. Simi- cells were activated in vitro by treatment with recombinant lar to the situation following estrogen treatment, expression Cdc25A and in vivo in cells overexpressing Cdc25A (Foster of c-Myc or cyclin D1 is sufficient to activate cyclin E-Cdk2 et al. 2001). These studies establish Cdc25A as another by promoting the formation of high molecular weight com- growth-promoting target of estrogen action and further indi- plexes lacking the CDK inhibitor p21WAF1/CIP1 (Prall et al. cate that estrogens independently regulate multiple compo- 1998, see Fig. 1). c-Myc expression was not accompanied by nents of the cell cycle machinery. increased cyclin D1 expression or Cdk4 activation, nor was There is compelling evidence that several mitogenic cyclin D1 induction accompanied by increases in c-Myc. growth factors, particularly those of the epidermal growth Similarly, others have demonstrated that activation of con- factor and insulin-like growth factor (IGF) families, interact ditional c-Myc alleles (MycER) does not activate cyclin D1 with ER-mediated signaling to regulate cell proliferation. transcription (Solomon et al. 1995) despite some conflicting Estrogens and insulin/IGF-I are potent mitogens for breast earlier reports (Jansen-Durr et al. 1993, Daksis et al. 1994). epithelial cells and, when co-administered, induce synergistic The ability of cyclin D1 induction to activate cyclin E-Cdk2 stimulation of cell proliferation (van der Burg et al. 1988). is compatible with the suggestion that synthesis of cyclin D1 In an MCF-7 breast cancer cell model where serum depri- is an important component of this response (Planas-Silva & vation and concurrent treatment with the antiestrogen ICI Weinberg 1997). However, an increase in cyclin D1 alone 182,780 inhibited both growth factor and estrogen action, co- is not sufficient to restore full activation of cyclin E-Cdk2 administration of insulin/IGF-I and estrogen induced syner- following growth arrest, unless cyclin D1 levels are elevated gistic stimulation of S-phase entry (Lai et al. 2001). This to approximately twofold the level induced by maximally was accompanied by synergistic activation of high molecular stimulatory concentrations of estrogen (Prall et al. 1998). In mass cyclin E–Cdk2 complexes lacking p21WAF1/CIP1. A cen- contrast, c-Myc induction to the level reached after estrogen tral component of this synergistic effect was the ability of treatment is sufficient to quantitatively mimic estrogen acti- estrogen to antagonize an insulin-induced increase in vation of cyclin E-Cdk2 and cell cycle progression. p21WAF1/CIP1 gene expression, with consequent activation of

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Figure 2 A model of antiestrogen effects on molecules regulating G1 phase progression in MCF-7 cells. See text for details. ICI, steroidal antiestrogen; D1, cyclin D1; E, cyclin E. Shaded cyclin – CDK complexes are inactive. cyclin E-Cdk2, again emphasising the importance of The importance of cyclin D1 in antiestrogen action is p21WAF1/CIP1 as an estrogen target. indicated by the observation that overexpression of cyclin D1 decreased sensitivity to antiestrogen inhibition at 24 and Molecular mechanisms of growth 48 h while overexpression of cyclin Eproduced a less pronounced early cell-cycle effect, indicating only partial resist- regulation by antiestrogens ance to antiestrogen inhibition in the short term (Hui et al. The pure antiestrogen ICI 182,780 arrests MCF-7 cells in a 2002). Neither overexpression of cyclin D1 or cyclin Econ- state with characteristics of quiescence (G0), as indicated by ferred antiestrogen resistance in longer-term assays (Pacilio the formation of p130/E2F4 complexes and the accumulation et al. 1998, Hui et al. 2002). p27Kip1 is another essential of hyperphosphorylated E2F4 (Carroll et al. 2000, Fig. 2). In mediator of cell cycle arrest by antiestrogens since antisense- this state they are relatively resistant to the mitogenic effects mediated down-regulation of p27Kip1 abrogates antiestrogen- of growth factors (Lai et al. 2001). Antiestrogen-mediated induced cell cycle arrest in MCF-7 human breast cancer cells

G0/G1 arrest is associated with decreased cyclin D1 gene (Cariou et al. 2000). Furthermore, MAP kinase (MAPK) acti- expression, inactivation of cyclin D1–Cdk4 complexes, and vation contributes to antiestrogen resistance through down- decreased phosphorylation of pRB (Musgrove et al. 1993, regulation of p27Kip1 (Donovan et al. 2001). However, Watts et al. 1995). Inhibition of cyclin E-Cdk2 activity also p21WAF1/CIP1 antisense treatment resulted in a decrease in occurs prior to a decrease in the S phase fraction, and is p27Kip1 protein levels, while changes in p21WAF1/CIP1 protein dependent on p21WAF1/CIP1 since treatment with antisense levels were not observed following treatment with p27Kip1 oligonucleotides to p21WAF1/CIP1 attenuates the effect (Carroll antisense (JS Carroll, A Swarbrick, EA Musgrove, & RL et al. 2000). Recruitment of p21WAF1/CIP1 to cyclin E–Cdk2 Sutherland, unpublished data). These data suggest that complexes is, in turn, dependent on decreased cyclin D1 p21WAF1/CIP1 initiates inhibition of cyclin E/Cdk2 and con- expression (Carroll et al. 2000, Fig. 2). sequent accumulation of p27Kip1. www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021183 01:29:17AM via free access Doisneau-Sixou et al.: Estrogen and antiestrogen action in breast cancer

Given the importance of c-Myc in estrogen-induced action, illustrated in Figs 1 and 2. This indicates that the mitogenesis, it is perhaps not surprising that constitutive mitogenic effects of estrogen appear to be mediated by at c-Myc expression confers resistance to antiestrogen treat- least two apparently distinct pathways, of which c-Myc and ment (Venditti et al. 2002). Oligonucleotide antisense inhi- cyclin D1, respectively, are the key regulators. The net result bition of c-Myc leads to inhibition of cyclin D1 expression, of estrogen-induced c-Myc or cyclin D1 expression is early subsequent redistribution of p21WAF1/CIP1 from cyclin D1– activation of cyclin E-Cdk2 by the formation of high molecu- Cdk4 to cyclin E–Cdk2 complexes, and a decline in cyclin lar weight cyclin E–Cdk2 complexes associated with the E-Cdk2 enzymatic activity (Carroll et al. 2002), thereby pocket protein p130 and deﬁcient in the CDK inhibitor recapitulating the initial downstream events that culminate in p21WAF1/CIP1. Phosphorylation of pRB is a primary target of growth arrest after ICI 182,780 treatment (Fig. 2). Simulta- cyclin E-Cdk2 activity, resulting in the well-documented neous repression of p21WAF1/CIP1 attenuated the growth- release of E2F transcription factors necessary for DNA syn- inhibitory effects of reduced c-Myc expression, emphasizing thesis and progression from G1 to S phase of the cell cycle. the importance of this CDK inhibitor in c-Myc action in these ‘Pure’ nonsteroidal antiestrogens, e.g. ICI 182,780, elicit cells (Carroll et al. 2002). While the effects of estrogens on a sequence of events that includes an acute decrease in cell cycle regulatory molecules largely mirror the effects of c-Myc, a subsequent decline in cyclin D1 and a consequent antiestrogens, one difference is the relationship between loss of cyclin D1–Cdk4 complexes. This results in redistri- c-Myc and cyclin D1. Induction of c-Myc does not increase bution of p21WAF1/CIP1 from cyclin D1–Cdk4/6 complexes to cyclin D1 expression, but antisense-mediated reduction in cyclin E–Cdk2 complexes, inhibition of cyclin E-Cdk2, c-Myc leads to decreased cyclin D1 expression. Thus, hypophosphorylation of the pocket proteins and ultimately although cyclin D1 expression is dependent on c-Myc, c-Myc growth arrest in a state with characteristics of quiescence is not limiting for cyclin D1 expression. (Fig. 2). Although the initial events in inhibition of cyclin Farnesyltransferase inhibitors (FTIs) are a new class of E-Cdk2 are predominantly mediated by p21WAF1/CIP1, p27Kip1 anticancer drugs that are presently in phase III clinical evalu- plays a major role in maintaining growth arrest. Some minor ation (Johnston & Kelland 2001). The demonstration that differences in response are apparent in different cell lines FTIs prevent estrogen-stimulated cell cycle progression (Watts et al. 1995, Carroll et al. 2000, Hui et al. 2002) but implicates prenylated proteins in estrogen action. Since the key features of this model, i.e. the central importance of major components of cytoplasmic signaling pathways c-Myc, cyclin D1 and redistribution of the CDK inhibitors include low molecular weight GTPases such as Ras that p21WAF1/CIP1 and p27Kip1, are conserved. One important but require prenylation for function, one mechanism for this underexplored issue is the degree to which the cell cycle effect is inhibition of the non-genomic effects of estrogen, events following treatment with the ‘pure’ steroidal antiestro- i.e. activation of the Src/MAPK/ERK pathway. Consistent gens are also elicited by selective estrogen modulators with this idea, FTIs interfere with estrogen regulation of pro- (SERMs) including nonsteroidal antiestrogens like tamox- gesterone receptor, cyclin D1 and c-Myc, but not genes con- ifen. There appears to be signiﬁcant overlap, but recent data taining a classical ERE (Doisneau-Sixou et al. 2003a). In from this laboratory indicate differences in the growth arrest MCF-7 cells, FTI L744,832 induces a two- to threefold states induced by the two classes of antiestrogen increase in cyclin D1 and markedly increases p21WAF1/CIP1 (unpublished data). transcription (Sepp-Lorenzino & Rosen 1998). This increase Emerging evidence indicates that the non-genomic in p21WAF1/CIP1 is p53-dependent and mediates the inhibition effects of estrogen play an important role in mediating its of cyclin E-Cdk2 activity, reduced pRB phosphorylation and mitogenic effects. The relative roles of the transcriptional

G1 arrest. FTI-277 and tamoxifen induce an additive effect activity of ERα and these non-genomic effects in mediating on inhibition of breast cancer cell proliferation and the addi- speciﬁc critical responses to estrogen, e.g. c-Myc and cyclin tive effect is probably predominantly due to the recruitment D1 induction, are as yet unclear, although recently developed of p27Kip1 and, to a less extent, p21WAF1/CIP1 into cyclin E– model systems offer new insights. For example, inhibition of Cdk2 complexes (Doisneau-Sixou et al., 2003b). By altering membrane ER signaling impairs estrogen activation of cyclin the non-genomic effects of ER, the FTIs may be a new class D1 and cell cycle progression (Razandi et al. 2003). Further of ‘antiestrogens’ to combine with tamoxifen or ICI 182,780. experimentation to explore such questions will offer important new insights into estrogen/antiestrogen action, with the Summary and conclusions possibility of identifying potential new therapeutic targets and prognostic indicators in breast cancer. The development of powerful in vitro model systems, wher- ein breast cancer cells are growth-arrested with a pure anti- Acknowledgements estrogen and cell cycle progression reinitiated with estrogen and/or overexpressed proteins, has facilitated dissection of Research in this laboratory is supported by the National some early molecular events in estrogen and antiestrogen Health and Medical Research Council (NHMRC) of

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