Cyclin D1 is necessary for tamoxifen-induced cell cycle progression in human breast cancer cells

Despite the success of tamoxifen in treating hormone-responsive breast cancer, its use is limited by the development of resistance to the drug. Understanding the pathways involved in the growth of tamoxifen-resistant cells may lead to new ways to treat tamoxifen-resistant breast cancer. Here, we investigate the role of cyclin D1, a mediator of estrogen-dependent proliferation, in growth of tamoxifen-resistant cells using a cell culture model of acquired resistance to tamoxifen. We show that tamoxifen and 4-hydroxytamoxifen (OHT) promoted cell cycle progression of tamoxifen-resistant cells after growth-arrest mediated by the estrogen receptor down-regulator ICI 182,780. Down-regulation of cyclin D1 with small interfering RNA blocked basal cell growth of tamoxifen-resistant cells and induction of cell proliferation by OHT. In addition, pharmacologic inhibition of phosphatidylinositol 3-kinase/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 pathways decreased basal cyclin D1 expression and impaired OHT-mediated cyclin D1 induction and cell cycle progression. These findings indicate that cyclin D1 expression is necessary for proliferation of tamoxifen-resistant cells and for tamoxifen-induced cell cycle progression. These results suggest that therapeutic strategies to block cyclin D1 expression or function may inhibit development and growth of tamoxifen-resistant tumors.     

Lapatinib antitumor activity is not dependent upon phosphatase and tensin homologue deleted on chromosome 10 in ErbB2-overexpressing breast cancers

Trastuzumab antitumor activity in ErbB2-overexpressing breast cancers seems to be dependent upon the presence of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a phosphatase that dampens phosphatidylinositol 3-kinase-Akt signaling. Consequently, PTEN deficiency, which occurs in 50% of breast cancers, predicts for resistance to trastuzumab monotherapy. Here, we show that lapatinib, a small-molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, exerts its antitumor activity in a PTEN-independent manner. Steady-state phosphorylated ErbB2 (p-ErbB2) and p-Akt (S473) protein levels were inhibited within 30 min following lapatinib but not in response to trastuzumab in BT474 and Au565 cells (two ErbB2-overexpressing breast cancer cell lines that are sensitive to the proapoptotic effects of lapatinib). Whereas trastuzumab reportedly inhibits SRC phosphorylation (Y416), which in turn reduced SRC-ErbB2 protein interactions, lapatinib had no effect on either variable. To assess the potential functional role that PTEN might play in lapatinib antitumor activity, we selectively knocked down PTEN in BT474 and Au565 cells using small interfering RNA transfection. Loss of PTEN did not affect induction of tumor cell apoptosis by lapatinib in either cell line. In addition, lapatinib inhibited Akt phosphorylation in MDA-MB-468 cells, an ErbB1-expressing/ErbB2 non-overexpressing breast cancer line, despite their PTEN-null status. Moreover, patients with ErbB2-overexpressing inflammatory breast cancers responded to lapatinib monotherapy regardless of PTEN status. Thus, lapatinib seems to exert its antitumor activity in ErbB2-overexpressing breast cancers in a PTEN-independent manner. These data emphasize the importance of assessing PTEN status in tumors when selecting ErbB2-targeted therapies in patients with breast cancer.     

Synergistic inhibition of breast cancer cell lines with a dual inhibitor of EGFR-HER-2/neu and a Bcl-2 inhibitor

The epidermal growth factor receptor (EGFR) (ErbB1) and HER-2/neu (ErbB2) are members of the ErbB family of receptor tyrosine kinases. These receptors are overexpressed in a variety of human tumors and overexpression generally correlates with poor prognosis and decreased survival. Lapatinib, a reversible inhibitor of both EGFR and HER-2/neu, has shown some success in achieving clinical responses in heavily pretreated advanced cancer patients. GW2974 is a reversible dual inhibitor similar to lapatinib, but GW2974 was not progressed to clinical trials due to pharmacokinetic issues. Bcl-2, an anti-apoptotic protein, is also overexpressed in a number of human tumors. Bcl-2 inhibitors induce apoptosis and sensitize cancer cells to other therapies. The purpose of this study was to assess the effects of combining ErbB and Bcl-2 inhibitors on the growth of human breast cancer cell lines. EGFR/HER-2/neu tyrosine kinase inhibitors (lapatinib and GW2974) were combined with Bcl-2 inhibitors (HA14-1 or GX15-070) and the anti-proliferative effects were determined by the MTT tetrazolium dye assay. Combinations were tested in MCF-7 human breast cancer cells, a HER-2/neu transfected MCF-7 cell line (MCF/18), and a tamoxifen-resistant MCF-7 cell line (MTR-3). A synergistic inhibitory effect was observed with the combination of inhibitors of EGFR-HER-2/neu (lapatinib or GW2974) and Bcl-2 (GX15-070 or HA14-1) on the growth of the MCF-7, MCF/18, and MTR-3 human breast cancer cell lines. This study suggests that simultaneously blocking the ErbB family of receptor tyrosine kinases and Bcl-2 family of proteins may be a benefit to breast cancer patients.     

Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells

Antibodies and small molecule tyrosine kinase inhibitors targeting ErbB2 exhibit distinct, noncross resistant mechanisms of action. Here, apoptosis of ErbB2-overexpressing breast cancer cells was enhanced by combining lapatinib, an inhibitor of ErbB1 and ErbB2 tyrosine kinases, with anti-ErbB2 antibodies, including (i) trastuzumab, a humanized monoclonal antibody, and (ii) pAb, rabbit polyclonal antisera generated by vaccination with a human ErbB2 fusion protein. Treating ErbB2-overexpressing breast cancer cell lines with a relatively low concentration of lapatinib alone resulted in a minimal increase in tumor cell apoptosis with an associated decrease in steady-state protein levels of p-ErbB2, p-Akt, p-Erk1/2, and notably survivin, compared to baseline. Exposure to pAb alone reduced total ErbB2 protein, disrupting ErbB3 transactivation, leading to a marked inhibition of p-Akt; however, survivin protein levels remained unchanged and apoptosis only increased slightly. Treatment with trastuzumab alone had relatively little effect on survivin and apoptosis was unaffected. Combining lapatinib with either pAb or trastuzumab markedly downregulated survivin protein and enhanced tumor cell apoptosis. The association between the inhibition of survivin and enhanced apoptosis following the combination of ErbB2-targeted therapies provides a biological effect in order to identify therapeutic strategies that promote tumor cell apoptosis and might improve clinical response.     

Estrogen receptor α is the major driving factor for growth in tamoxifen-resistant breast cancer and supported by HER/ERK signaling

Resistance to tamoxifen is a major clinical challenge in the treatment of breast cancer; however, it is still unclear which signaling pathways are the major drivers of tamoxifen-resistant growth. To characterize resistance mechanisms, we have generated different tamoxifen-resistant breast cancer cell lines from MCF-7. In this model, we investigated whether signaling from human epidermal growth factor receptors (HERs), their downstream kinases, or from the estrogen receptor α (ERα) was driving tamoxifen-resistant cell growth. Increased expression of EGFR and increased phosphorylation of HER3 were observed upon acquisition of tamoxifen resistance, and the extracellular activated kinase (ERK) signaling pathway was highly activated in the resistant cells. The EGFR inhibitor gefitinib and the ERK pathway inhibitor U0126 resulted in partial and preferential growth inhibition of tamoxifen-resistant cells. All the tamoxifen-resistant cell lines retained ERα expression but at a lower level compared to that in MCF-7. Importantly, we showed via ERα knockdown that the tamoxifen-resistant cells were dependent on functional ERα for growth and we observed a clear growth stimulation of resistant cell lines with clinically relevant concentrations of tamoxifen and 4-OH-tamoxifen, indicating that tamoxifen-resistant cells utilize agonistic ERα stimulation by tamoxifen for growth. The tamoxifen-resistant cells displayed high phosphorylation of ERα at Ser118 in the presence of tamoxifen; however, treatment with U0126 neither affected the level of Ser118 phosphorylation nor expression of the ERα target Bcl-2, suggesting that ERK contributes to cell growth independently of ERα in our cell model. In support of this, combined treatment against ERα and ERK signaling in resistant cells was superior to single-agent treatment and as effective as fulvestrant treatment of MCF-7 cells. Together, these findings demonstrate that ERα is a major driver of growth in tamoxifen-resistant cells supported by HER/ERK growth signaling, implying that combined targeting of these pathways may have a clinical potential for overcoming tamoxifen resistance.     

ErbB2-overexpressing human mammary carcinoma cells display an increased requirement for the phosphatidylinositol 3-kinase signaling pathway in anchorage-independent growth.

The proto-oncogene ErbB2 is known to be amplified and to play an important role in the development of about one-third of human breast cancers. Phosphatidylinositol 3-kinase (PI3K), which is often activated in ErbB2-overexpressing breast cancer cells, is known to regulate cell proliferation and cell survival. Selective inhibitors of the PI3K pathway were used to assess the relevance of PI3K signaling in the anchorage-independent growth of a series of human mammary carcinoma cell lines. Wortmannin, LY294002, and rapamycin at concentrations that did not affect MAPK phosphorylation but substantially inhibited PI3K, Akt, and p70(S6K) significantly suppressed the soft agar growth of tumor cell lines that overexpress ErbB2 but not the growth of tumor lines with low ErbB2 expression. A similar growth inhibition of ErbB2-overexpressing carcinoma lines was observed when a dominant negative p85(PI3K) mutant was introduced into these cells. Forced expression of ErbB2 in breast cancer lines originally expressing low ErbB2 levels augmented receptor expression and sensitized those lines to LY294002- and rapamycin-mediated inhibition of colony formation. Furthermore, treatment with LY294002 resulted in the selective increase of cyclin-dependent kinase inhibitors p21(Cip1) or p27(Kip1) and suppression of cyclin E-associated Cdk2 kinase activity in ErbB2-overexpressing lines, which may account for their hypersensitivity toward inhibitors of the PI3K pathway in anchorage-independent growth. Our results indicate that the PI3K/Akt/p70(S6K) pathway plays an enhanced role in the anchorage-independent growth of ErbB2-overexpressing breast cancer cells, therefore providing a molecular basis for the selective targeting of this signaling pathway in the treatment of ErbB2-related human breast malignancies.     

Inhibition of heregulin mediated MCF-7 breast cancer cell growth by the ErbB3 binding protein EBP1

The ErbB2/3 heterodimer plays a critical role in breast cancer genesis and progression. EBP1, an ErbB3 binding protein, inhibits breast cancer growth but its effects on ErbB3 ligand mediated signal transduction or ErbB receptors is not known. We report here that ectopic expression of EBP1 in MCF-7 and AU565 breast cancer cell lines inhibited HRG-induced proliferation. ErbB2 protein levels were substantially decreased in EBP1 transfectants, while ErbB3 levels were unchanged. HRG-induced AKT activation was attenuated in EBP1 stable transfectants and transfection of a constitutively activated AKT partially restored the growth response to HRG. Down-regulation of EBP1 expression in MCF-7 cells by shRNA resulted in increased cell growth in response to HRG and increased cyclin D1 and ErbB2 expression. These results suggest that EBP1, by down-regulating ErbB signal transduction, attentuates HRG-mediated growth of breast cancer cells.     

Tamoxifen resistance in breast tumors is driven by growth factor receptor signaling with repression of classic estrogen receptor genomic function

Not all breast cancers respond to tamoxifen, and many develop resistance despite initial benefit. We used an in vivo model of estrogen receptor (ER)-positive breast cancer (MCF-7 xenografts) to investigate mechanisms of this resistance and develop strategies to circumvent it. Epidermal growth factor receptor (EGFR) and HER2, which were barely detected in control estrogen-treated tumors, increased slightly with tamoxifen and were markedly increased when tumors became resistant. Gefitinib, which inhibits EGFR/HER2, improved the antitumor effect of tamoxifen and delayed acquired resistance, but had no effect on estrogen-stimulated growth. Phosphorylated levels of p42/44 and p38 mitogen-activated protein kinases (both downstream of EGFR/HER2) were increased in the tamoxifen-resistant tumors and were suppressed by gefitinib. There was no apparent increase in phosphorylated AKT (also downstream of EGFR/HER2) in resistant tumors, but it was nonetheless suppressed by gefitinib. Phosphorylated insulin-like growth factor-IR (IGF-IR), which can interact with both EGFR and membrane ER, was elevated in the tamoxifen-resistant tumors compared with the sensitive group. However, ER-regulated gene products, including total IGF-IR itself and progesterone receptor, remained suppressed even at the time of acquired resistance. Tamoxifen's antagonism of classic ER genomic function was retained in these resistant tumors and even in tumors that overexpress HER2 (MCF-7 HER2/18) and are de novo tamoxifen-resistant. In conclusion, EGFR/HER2 may mediate tamoxifen resistance in ER-positive breast cancer despite continued suppression of ER genomic function by tamoxifen. IGF-IR expression remains dependent on ER but is activated in the tamoxifen-resistant tumors. This study provides a rationale to combine HER inhibitors with tamoxifen in clinical studies, even in tumors that do not initially overexpress EGFR/HER2.     

p27(Kip1) induces quiescence and growth factor insensitivity in tamoxifen-treated breast cancer cells

Tamoxifen, a selective estrogen-receptor modulator, is effective in the treatment and prevention of breast cancer, but therapeutic resistance is common. Pure steroidal antiestrogens are efficacious in tamoxifen-resistant disease and, unlike tamoxifen, arrest cells in a state of quiescence from which they cannot reenter the cell cycle after growth factor stimulation. We now show that in hydroxytamoxifen-treated cells, transduction of the cell cycle inhibitor p27(Kip1) induces quiescence and insensitivity to growth stimulation by insulin/insulin-like growth factor I and epidermal growth factor/transforming growth factor alpha. Furthermore, reinitiation of cell cycle progression by insulin/insulin-like growth factor I in hydroxytamoxifen-arrested cells involves dissociation of the corepressors nuclear receptor corepressor (N-CoR) and silencing mediator for retinoid and thyroid hormone receptor (SMRT) from nuclear estrogen receptor alpha and redistribution to the cytoplasm, a process that is inhibited by mitogen-activated protein/extracellular signal-regulated kinase, but not phosphatidylinositol 3'-kinase, inhibitors. These data suggest that agents that up-regulate p27(Kip1) or inhibit growth factor signaling via the extracellular signal-regulated kinases should be tested as therapeutic strategies in tamoxifen-resistant breast cancer.     

Controlled activation of ErbB1/ErbB2 heterodimers promote invasion of three-dimensional organized epithelia in an ErbB1-dependent manner: implications for progression of ErbB2-overexpressing tumors

Receptor tyrosine kinases of the ErbB family are implicated in a number of cancers, including that of the breast. ErbB receptors are activated by ligand-induced formation of homodimers and heterodimers. Receptor heterodimerization is thought to play a critical role in breast cancers overexpressing multiple members of the ErbB family. Although coexpression of ErbB receptors is associated with poor patient prognosis, the mechanisms by which receptor heterodimerization regulates tumor progression are not clear, due in part to a lack of methods that allow controlled activation of specific receptor heterodimers in mammary epithelial cells. Here, we report an approach to activate ErbB1-ErbB2 heterodimers in a nontumorigenic breast epithelial cell line, MCF-10A, without interference from endogenous ErbB receptors. Using such a method, we show that whereas both ErbB2 homodimers and ErbB1-ErbB2 heterodimers were equally potent in activating the Ras/mitogen-activated protein kinase pathway, the heterodimers were more potent in activating the phosphoinositide 3'-kinase (PI3K) and phospholipase Cgamma1 pathways than ErbB2 homodimers. We combined the dimerization system with a three-dimensional cell culture approach to show that whereas both ErbB2 homodimers and ErbB1-ErbB2 heterodimers induced disruption of three-dimensional acini-like structures, only heterodimers promoted invasion of cells through extracellular matrix. The ability of heterodimers to induce invasion required the ErbB1 kinase activity and required activation of PI3K, Ras/mitogen-activated protein kinase, and phospholipase Cgamma1 signaling pathways. Thus, we have identified cell invasion as a heterodimer-specific biological outcome and suggest that coexpression of ErbB1 may critically regulate invasive progression of ErbB2-positive breast cancers.     

A lipid-soluble iron chelator alters cell cycle regulatory protein binding in breast cancer cells compared to normal breast cells

The lipid-soluble iron chelator desferri-exochelin (D-Exo) causes reversible cell cycle arrest in normal human mammary epithelial cells (NHMEC) but triggers apoptotic cell death in human breast cancer cells. We studied the effects of iron chelation with D-Exo on cell cycle regulatory proteins in cultures of NHMEC and MCF-7 breast cancer cells. In co-immunoprecipitation studies, D-Exo inhibited binding of cyclins A and E to cyclin dependent kinase 2 (CDK2) in NHMEC, but in MCF-7 cells binding of these cyclins to CDK2 was enhanced. D-Exo treatment markedly increased expression of p53 and increased binding of p21 to CDK2 in the MCF-7 cells but not in NHMEC. Therefore differences in effects of iron chelation on cell cycle protein binding in cancer cells compared to normal cells may trigger apoptosis in cancer cells while normal breast cells are spared.     

Study of the biologic effects of lapatinib, a reversible inhibitor of ErbB1 and ErbB2 tyrosine kinases, on tumor growth and survival pathways in patients with advanced malignancies.

PURPOSE: This was a pilot study to assess the biologic effects of lapatinib on various tumor growth/survival pathways in patients with advanced ErbB1 and/or ErbB2-overexpressing solid malignancies. PATIENTS AND METHODS: Heavily pretreated patients with metastatic cancers overexpressing ErbB2 and/or expressing ErbB1 were randomly assigned to one of five dose cohorts of lapatinib (GW572016) administered orally once daily continuously. The biologic effects of lapatinib on tumor growth and survival pathways were assessed in tumor biopsies obtained before and after 21 days of therapy. Clinical response was determined at 8 weeks. RESULTS: Sequential tumor biopsies from 33 patients were examined. Partial responses occurred in four patients with breast cancer, and disease stabilization occurred in 11 others with various malignancies. Responders exhibited variable levels of inhibition of p-ErbB1, p-ErbB2, p-Erk1/2, p-Akt, cyclin D1, and transforming growth factor alpha. Even some nonresponders demonstrated varying degrees of biomarker inhibition. Increased tumor cell apoptosis (TUNEL) occurred in patients with evidence of tumor regression but not in nonresponders (progressive disease). Clinical response was associated with a pretreatment TUNEL score > 0 and increased pretreatment expression of ErbB2, p-ErbB2, Erk1/2, p-Erk1/2, insulin-like growth factor receptor-1, p70 S6 kinase, and transforming growth factor alpha compared with nonresponders. CONCLUSION: Lapatinib exhibited preliminary evidence of biologic and clinical activity in ErbB1 and/or ErbB2-overexpressing tumors. However, the limited sample size of this study and the variability of the biologic endpoints suggest that further work is needed to prioritize biomarkers for disease-directed studies, and underscores the need for improved trial design strategies in early clinical studies of targeted agents.     

Increased Constitutive Activity of PKB/Akt in Tamoxifen Resistant Breast Cancer MCF-7 Cells

The tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell line has been used as a model to identify the signalling pathways that enable resistant cancer cells to grow independently of steroid hormones. In TAM-R cells, peptide growth factor signalling pathways appear to be important in modified cell behaviour, growth and survival. The PI3 kinase signalling components Akt1 and Akt2 are expressed at similar levels by both parental wild-type MCF-7 and TAM-R cells, but Akt1 phosphorylation is significantly increased in TAM-R cells grown under basal conditions. High levels of basal Akt, GSK3 alpha / beta and p70S6 kinase phosphorylation are all inhibited by the PI3 kinase inhibitor, LY 294002. The ligands for the EGFR/erbB1 receptor, EGF (epidermal growth factor) and TGF alpha (transforming growth factor- alpha ) demonstrate an increased ability to activate Akt in TAM-R compared with parental MCF-7 cells and it is proposed that the preferred autocrine or paracrine activation of Akt occurs via the erbB heterodimer EGFR/erbB2 in TAM-R cells. Akt phosphorylation is reduced by gefitinib ("Iressa"/ZD1839). The results suggest that the PI3 kinase pathway plays a role in proliferation of TAM-R cells and is important in the increased EGF induced membrane ruffling detected in the resistant cells. Increased Akt1 activation may contribute to the aggressive phenotype of tamoxifen resistant ER (oestrogen receptor) positive breast cancers.     

Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells

The ErbB2 receptor tyrosine kinase is overexpressed in a variety of human tumours. In order to understand the mechanism by which ErbB2 mediates tumour proliferation we have functionally inactivated the receptor using an intracellularly expressed, ER-targeted single-chain antibody (scFV-5R). Inducible expression of scFv-5R in the ErbB2-overexpressing SKBr3 breast tumour cell line leads to loss of plasma membrane localized ErbB2. Simultaneously, the activity of ErbB3, MAP kinase and PKB/Akt decreased dramatically, suggesting that active ErbB2/ErbB3 dimers are necessary for sustained activity of these kinases. Loss of functional ErbB2 caused the SKBr3 tumour cells to accumulate in the G1 phase of the cell cycle. This was a result of reduction in CDK2 activity, which was mediated by a re-distribution of p27Kip1 from sequestering complexes to cyclin E/CDK2 complexes. The level of c-Myc and D-cyclins, proteins involved in p27KiP1 sequestration, decreased in the absence of functional ErbB2. Ectopic expression of c-Myc led to an increase in D cyclin levels, CDK2 activity and resulted in a partial G1 rescue. We propose that c-Myc is a primary effector of ErbB2-mediated oncogenicity and functions to prevent normal p27Kip1 control of cyclinE/CDK2.     

Role of cyclin D1 in ErbB2-positive breast cancer and tamoxifen resistance

Cyclin D1 plays an important role in the regulation of the G1 phase in the cell cycle. In mammary epithelial cells the expression of cyclin D1 is regulated through the oestrogen receptor and via ErbB2 signalling. Here we investigated the prognostic significance of cyclin D1 among 230 breast cancer patients randomised for tamoxifen, CMF chemotherapy and radiotherapy. The importance of combined cyclin D1 and ErbB2 overexpression was also analysed. Immunohistochemical analysis of the cyclin D1 expression resulted in 69 (29.8%) weakly positive, 107 (46.5%) moderately positive and 54 (23.7%) strongly positive cases. The prognostic importance of ErbB2 was significantly greater for patients whose tumours overexpressed cyclin D1 than for other patients (p = 0.026). In the former group, ErbB2 overexpression was strongly associated with increased risk of recurrence (RR = 4.7; 95% CI, 2.1–10.4) and breast cancer death (RR = 5.4; 95% CI, 2.3–12.6). This result is in accordance with experimental studies demonstrating a link between cyclin D1 and ErbB2 in oncogenesis. Among oestrogen receptor positive patients, those with moderate cyclin D1 expression significantly did benefit from tamoxifen treatment (RR = 0.42; 95% CI, 0.21–0.82) whereas those with weak or strong expression did not. Therefore cyclin D1 might be a predictive marker for tamoxifen resistance.     

Mechanisms of growth arrest by c-myc antisense oligonucleotides in MCF-7 breast cancer cells: implications for the antiproliferative effects of antiestrogens

The proto-oncogene c-myc is up-regulated by estrogen stimulation of hormone-dependent breast cancer cells and is frequently overexpressed in breast and other cancers. Therapeutic interventions that inhibit c-Myc expression have been extensively investigated, including antisense oligonucleotides that have high specificity and potential clinical application. This investigation compared antiestrogen-mediated growth arrest with the molecular events after repression of c-Myc expression in MCF-7 breast cancer cells using an antisense oligonucleotide. We show that the decreased cellular proliferation of MCF-7 cells after direct inhibition of c-Myc is a consequence of inhibition of cyclin D1 expression, subsequent redistribution of p21(WAF1/CIP1) from cyclin D1-Cdk4 to cyclin E-Cdk2 complexes, and a decline in cyclin E-Cdk2 enzymatic activity. Simultaneous repression of p21(WAF1/CIP1) can attenuate the growth-inhibitory effects of reduced c-Myc expression emphasizing the importance of this cyclin-dependent kinase (CDK) inhibitor in growth arrest. These molecular events are similar to the initial changes in cyclin gene expression, CDK complex formation and CDK activity seen after antiestrogen (ICI 182780)-mediated growth inhibition of MCF-7 cells, which suggests that the down-regulation of c-Myc by ICI 182780 is a primary event that culminates in cell cycle arrest.