Constitutive Overexpression of Cyclin D1 but not Cyclin E Confers Acute Resistance to Antiestrogens in T-47D Breast Cancer Cells¹

Breast cancer is a heterogenous disease with regard to its morphology, invasive behavior, metastatic capacity, hormone receptor expression, responsiveness to treatment, and clinical outcome. Both ER³ and PR are used routinely in the clinical management of breast cancer as predictors of a patient response to endocrine therapy, and as weak prognostic indicators of the clinical course of a patient. Two-thirds of all breast cancers are ER-positive, and ER is a molecular target for endocrine therapy. The nonsteroidal antiestrogen tamoxifen is the endocrine treatment of choice and is used with or without chemotherapy in the management of all stages of ER-positive breast cancer in both pre- and postmenopausal women. However, only ∼50% of patients with ER-positive tumors and ∼75% of patients with tumors exhibiting both ER and PR positivity will respond to endocrine therapy. Moreover, acquired resistance is a major problem in breast cancer management and, thus, tamoxifen may only be effective for a limited period.

Cancer is a genetic disease where successive mutations lead to the progressive loss of normal homeostatic mechanisms that control cell proliferation, differentiation, and death, giving the cell a selective advantage in its environment and leading to clonal expansion. Normal cell proliferation is under strict regulation. There is a physiological restriction point late in G₁ where the cell integrates signals it receives from the internal and external environment, and commits itself to passage from G₁ phase to S phase. Beyond this checkpoint, the cell becomes refractory to the effects of external growth stimuli and hormonal influences, and is destined to DNA replication and ultimately cell division (1). Estrogen and antiestrogens exert their effects in the G₁ phase of the cell cycle, promoting or inhibiting cell cycle progression. Cyclins belonging to the D and E families, and their respective kinase partners, Cdk4/6 and Cdk2, are involved in late G₁ restriction point control (2, 3). Deregulated expression of cyclin D1 or cyclin E renders growth of normal cells less dependent on growth factors and accelerates passage through G₁ phase of the cell cycle (4). Overexpression of either cyclin D1 or cyclin E leads to mammary carcinoma in transgenic mice, suggesting roles as oncogenes in mammary epithelium (5, 6). Moreover, both cyclin D1 and cyclin E are overexpressed in a substantial proportion of breast cancers, 45% and 30%, respectively (7, 8, 9), and some studies have indicated that overexpression of either gene is associated with poor prognosis in breast cancer (8, 10), although other studies have failed to demonstrate such relationships (11, 12, 13, 14).

There is now general consensus that cyclin D1 abundance is positively correlated with ER positivity in breast cancer (13, 14, 15). Our earlier studies indicated that both CCND1 amplification and cyclin D1 mRNA overexpression are associated with poor prognosis in ER-positive breast cancer patients (10, 16). One mechanism by which overexpression of cyclin D1 may lead to a worse clinical outcome is by conferring resistance to endocrine treatment. Consistent with this possibility, a recent clinical study from this laboratory suggested that the duration of the response to tamoxifen was significantly longer in ER-positive patients with low cyclin D1 mRNA levels than in those with high cyclin D1 (10), although these analyses must be interpreted with caution because of the small sample size. Additional indirect support for this hypothesis comes from previous in vitro studies demonstrating that a reduction in cyclin D1 mRNA and protein expression is an early and critical event in antiestrogen action (17, 18). In addition, short-term ectopic induction of cyclin D1 expression in ER-positive breast cancer cell lines (T-47D and MCF-7) can overcome the inhibition of cell cycle progression induced by antiestrogen (19). Together these data suggested that overexpression of cyclin D1 in ER-positive tumors may lead to insensitivity to antiestrogens. However, a study published more recently indicated that inducible cyclin D1 overexpression in MCF-7 breast cancer cells does not prevent inhibition of cell growth by antiestrogens (20).

Patients with breast cancer overexpressing cyclin E have a significantly increased risk of relapse and death (8, 21). About 40% of breast cancers overexpressing cyclin E have mutant pRb and high p16^INK4a expression suggesting that abnormal cyclin E expression may be linked to deregulation of the cyclin D1-Cdk4-p16^INK4a-pRb pathway (22). Given that cyclin E can functionally replace cyclin D1 in mice (23), overexpression of cyclin E may have effects similar to overexpression of cyclin D1, but the role of cyclin E as a marker of therapeutic responsiveness to antiestrogens had not been elucidated when this study was initiated. A recent publication addressing this question has indicated that overexpression of cyclin E in MCF-7 cells leads to partial resistance to 48-h tamoxifen treatment (24).

Given that published data provide preliminary evidence that levels of cyclin D1 and cyclin E expression may influence therapeutic sensitivity to antiestrogens, we investigated this hypothesis in vitro using clonal T-47D breast cancer cell lines constitutively overexpressing either cyclin D1 or cyclin E.

The cell lines derived from T-47D human breast cancer cells were cultured in RPMI 1640 supplemented with 10% FCS and insulin (10 μg/ml). For experiments investigating the short-term effects of ICI 182780 150-cm² flasks were seeded with 4 × 10⁶ cells. ICI 182780 {7α-[9-(4,4,5,5,5-pentafluropentylsulfinyl) nonyl]estra-1,3,5,(10)-triene-3,17β-diol, a kind gift from Dr. Alan Wakeling, Astra Zeneca Pharmaceuticals, Alderley Park, Cheshire, United Kingdom} was dissolved in ethanol to 10⁻² m. The final concentration of ethanol in the tissue culture medium was <0.07% and had no effect on the rate of cell proliferation. At the completion of experiments cells were harvested by brief incubation with trypsin (0.05% w/v)/EDTA (0.02% w/v) as described previously (25) or as described below. Cell cycle phase distribution was determined by analytical DNA flow cytometry as described previously (26).

The T-47D line from the E. G. and G. Mason Research Institute (Worcester, MA) was cloned by limiting dilution, and one clonal cell line, T-47D (7-2), was selected for transfection studies (27). T-47D (7-2) retained the characteristics of the parent line by all of the tested criteria, in particular sensitivity to growth regulation by steroids and steroid antagonists and abundance of cyclin D1 mRNA. A clonal cell line, Clone 17, was established by transfection of T-47D (7-2) breast cancer cells with the Tet-responsive transcriptional activator containing the wild-type Tet repressor and the VP16 activation domain of herpes simplex virus. Additional clonal cell lines were established by transfection of Clone 17 cells with empty tetracycline-repressed pTRE vector, or full-length cyclin D1 or cyclin E in the pTRE (Tet-responsive element) vector (Clontech Laboratories, Palo Alto, CA). Electroporation was carried out in a Bio-Rad Gene Pulser at 950 μF and 0.22 kV/cm. pTK-Hyg was cotransfected into the cells with each gene construct of interest providing a selectable marker. Twenty stable clones transfected with the cyclin D1 construct, 34 clones transfected with the full-length cyclin E construct, and 3 clones transfected with the empty pTRE vector were isolated.

Cells were lysed as follows: T-47D cell monolayers were washed twice in ice-cold PBS then scraped into ice-cold lysis buffer [50 mm HEPES (pH 7.5), 150 mm NaCl, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mm MgCl₂, 1 mm EGTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 200 μm sodium orthovanadate, 10 mm PP_i, 100 mm NaF, and 1 mm DTT]. The lysates were incubated for 5 min on ice and the cellular debris cleared by centrifugation (15,000 × g, 5 min, 4°C). Equal amounts of total protein (20–40 μg) were separated by SDS-PAGE then transferred to nitrocellulose filters. Proteins were visualized using the enhanced chemiluminescence detection system (Amersham, Castle Hill, Australia) after incubation (2 h at room temperature or overnight at 4°C) with the following primary antibodies: cyclin D1 (DCS-6) from Novacastra Laboratories Ltd., Newcastle-upon-Tyne, United Kingdom; cyclin E (C-19) from Santa Cruz Biotechnology Inc., Santa Cruz, CA; pRb (G3-245) from PharMingen, San Diego, CA; p21 from Transduction Laboratories, Lexington, KY; or p27 (Transduction Laboratories).

For assessment of cyclin E-associated kinase activity, cell monolayers were washed twice with PBS then scraped into 1 ml of ice-cold lysis buffer. The lysate was vortexed and placed on ice for 5–10 min, then centrifuged at 15,000 × g for 5 min at 4°C and the supernatant stored at −80°C. Cyclin E complexes were immunoprecipitated from equivalent amounts of protein with rabbit polyclonal antihuman cyclin E antiserum conjugated to protein A-Sepharose for 2 h at 4°C (PharMingen). The immunoprecipitates were washed twice with ice-cold 50 mm HEPES (pH 7.5) and 1 mm DTT.

The kinase reactions were initiated by resuspending the beads in 30 μl kinase buffer [50 mm HEPES (pH 7.5), 1 mm DTT, 2.5 mm EGTA, 10 mm MgCl₂, 20 mm ATP, 10 μCi [γ-³²P]ATP, 0.1 mm orthovanadate, 1 mm NaF, and 10 mm β-glycerophosphate] containing 10 μg histone H1 as a substrate. After incubation for 15 min at 30°C the reactions were terminated by the addition of 10 μl of 3× SDS sample buffer [187 mm Tris-HCl (pH 6.8), 30% (v/v) glycerol, 6% SDS, and 15% (v/v) β-mercaptoethanol]. The samples were then incubated at 95°C for 2 min, separated using 10% SDS-PAGE, and the dried gel exposed to X-ray film. Relative band intensities were quantitated by densitometric analysis (Molecular Dynamics, Sunnyvale, CA). Quantitation of protein levels by this method was linear over the range of protein concentrations and exposure times used in these studies.

Immunoprecipitation of p21 and p27 was performed using the method described above (for immunoprecipitating cyclin E for kinase activity assays), except that the antibodies were chemically cross-linked to protein A-Sepharose to reduce background (28). Antibodies used were rabbit polyclonal antibodies to human p21 (Santa Cruz Biotechnology Inc.; C-19) and human p27 (Santa Cruz Biotechnology Inc.; C-19).

The immunoprecipitated proteins were resuspended in 1× SDS sample buffer, separated by SDS-PAGE, transferred to nitrocellulose membrane, and the proteins detected using the antibodies described for Western blotting above.

Cell viability after drug treatment was assessed in a colony-forming assay. After harvest from the monolayer, cells were counted, and the appropriate dilutions were made with medium containing the supplements listed above and 5% FCS. The desired number of cells (normally 5 × 10³) was plated into duplicate 6-cm plates in 6 ml of medium. The dishes were placed in 37°C incubators with 95% air-5% CO₂ for 21 days.

After incubation, the medium was removed, and the cells were fixed and stained using the DIFF-Quik STAIN SET 64851 (Lab Aids Pty. Ltd., Narrabeen, Australia). The number of macroscopic colonies were counted using Quantity One 4.2.1 (Bio-Rad Laboratories, Hercules, CA).

[³⁵S]methionine/cysteine pulse-chase analysis was used to assess the half-life of cyclin D1 protein. Cell monolayers were washed once with methionine- and cysteine-free RPMI 1640 containing 5% (v/v) FCS, l-glutamine (6 mm), and insulin (10 μg/ml), and then incubated (the “pulse”) in methionine- and cysteine-free RPMI 1640 containing 200–300 μCi/ml [³⁵S]methionine and cysteine (Trans ³⁵S label; ICN) for 30 min. The [³⁵S]methionine/cysteine-containing cell culture medium was then decanted from the cell monolayer and replaced with unlabeled RPMI 1640 containing 5% (v/v) FCS after one wash in the same medium. Cells that had been treated with ICI 182780 were washed and cultured in medium containing the same levels of ICI 182780. After labeling, cells were harvested at various intervals (the “chase”) for immunoprecipitation using cyclin D1 antiserum and SDS-PAGE as described above.

A 15mer p27^Kip1 antisense oligonucleotide (29) was synthesized (Geneworks, Adelaide, SA, Australia) with phosphorothioate residues at the 5′ and 3′ terminal. A complementary (sense) oligonucleotide was also manufactured. Cyclin E-overexpressing (E 17–2) cells were harvested, gently syringed four times to minimize clumps, and 5 × 10⁵ cells were grown in 50-cm² dishes overnight. Twenty μl of Cellfectin (Life Technologies, Inc., Grand Island, NY) and oligonucleotide (2 μm final concentration) were incubated in 1 ml of serum-free RPMI 1640 for 30 min and subsequently added to the monolayer with 1 ml of RPMI 1640 supplemented with 10% FCS. All of the control oligonucleotides were included at 2 μm final concentration. The dishes were placed in a 37°C incubator with 5% CO₂ for 2–3 h with intermittent mixing. The oligonucleotide/Cellfectin solution was then decanted and the monolayer washed once with RPMI 1640 (5% FCS). Ten ml of RPMI 1640 (5% FCS) was then added to each dish. Cells were harvested for protein analysis as described above.

To address whether cyclin D1 and cyclin E are predictive markers for therapeutic responsiveness to antiestrogens in breast cancer, cell lines overexpressing cyclin D1 or cyclin E were produced using a tetracycline-controlled gene expression system. Two clones overexpressed cyclin E and two overexpressed cyclin D1 in the absence of tetracycline. Although cyclin E expression in clone E 17-3 could be repressed by a low concentration of tetracycline (2 μg/ml), the expression of cyclin E in the other clone E 17-2 could not be repressed at all by tetracycline. Repression of cyclin D1 expression was only achievable with high concentrations of tetracycline (10–15 μg/ml) in the two clones that overexpressed cyclin D1. To avoid the cytotoxic effect of tetracycline, the clones transfected with empty vector were used as control in preference to tetracycline-mediated repression. Western analysis demonstrated that in the absence of tetracycline, clone D1 17-1 constitutively overexpressed cyclin D1 protein by 5-fold, whereas clones E 17-2 and E 17-3 constitutively overexpressed cyclin E by 6- and 3-fold, respectively (Fig. 1 A). These T-47D breast cancer cells overexpressed cyclin D1 or cyclin E at levels similar to those reported previously for human breast cancer (7) and were therefore selected for additional study.

Inhibition of cyclin D1 gene expression with concurrent decline in cyclin D1 mRNA and protein levels is an early and critical event in antiestrogen action after acute (0–48 h) treatment of T-47D and MCF-7 breast cancer cells with antiestrogens (17, 18, 30). Therefore, we first tested the effects of the pure steroidal antiestrogen ICI 182780 on the abundance of cyclin D1 or cyclin E protein in the transfected cell lines. In the empty vector cells, cyclin D1 gene expression was down-regulated by ICI 182780 (Fig. 1,B). In the cyclin D1-overexpressing cell line, cyclin D1 expression was maintained for at least 72 h after treatment. Cyclin E levels appeared to be slightly reduced by ICI 182780 in the empty vector cells but increased slightly 24 h after ICI 182780 treatment in both cyclin E-overexpressing cell lines (Fig. 1 B).

The S phase fraction was measured using flow cytometry to assess whether overexpression of cyclin D1 or cyclin E provided any short-term proliferative advantage to cells treated with ICI 182780. Treatment of the cyclin D1-overexpressing cell line led to substantial resistance to antiestrogenic effects on cell cycle progression at 24 h. The E 17-2 cells demonstrated a smaller effect at 24 h but were still less sensitive than control cells (Fig. 2,A). With more extended treatment, both cyclin-overexpressing cell lines were increasingly inhibited and by 72 h approached the sensitivity of the control cell line (Fig. 2, B and C).

Because both cyclin D1 and cyclin E direct the kinase activity of their associated Cdks in phosphorylation of substrates including pRb, the phosphorylation state of the retinoblastoma protein pRb and the Cdk activities are important indicators of cell cycle progression. The hypophosphorylated (faster mobility) form of pRb predominated after 24 h of treatment of the empty vector cells with ICI 182780 in accordance with the inhibition of cell proliferation. The abundance of pRb in the empty vector cells was reduced significantly after 48 h of antiestrogen treatment. In contrast, hyperphosphorylated pRb remained abundant after treatment of the cyclin D1-overexpressing cell line D1 17-1, consistent with resistance to early cell cycle inhibition. The E 17-2 cell line displayed an intermediate effect, with some reduction in pRb phosphorylation (Fig. 3,A). The E 17-3 cell line, expressing a lower level of cyclin E, showed a similar effect to the empty vector cell line, although hyperphosphorylated pRb was still evident at 48 h. These findings are consistent with the results obtained from measurement of S phase fraction (Fig. 2).

The kinase activities of cyclin E-Cdk2 and cyclin D1-Cdk4 were next examined. The abundance of Cdk4-phosphorylated pRb was maintained in the D1 17-1 cells, but reduced in the empty vector and E 17-2 cells after treatment with ICI 182780, indicating that cyclin D1-Cdk4 kinase activity is maintained by overexpression of cyclin D1 (Fig. 3,B). Cyclin E-Cdk2 activity decreased in both the empty vector cells and the E 17-2 cells after ICI 182780 treatment. However, the activity was maintained slightly longer in the E 17-2 cells compared with the empty vector cells. The cyclin E-Cdk2 kinase activity was maintained in the D1 17-1 cells for at least 24 h after ICI 182780 treatment (Fig. 3,B), consistent with continued cell proliferation as evidenced from data on pRb phosphorylation and S phase fraction (Fig. 2; Fig. 3 A).

A recent study from our laboratory demonstrated that there is a substantial increase in the amount of cyclin E-associated p21 and p27 in MCF-7 breast cancer cells after antiestrogen treatment, and the initial decline in cyclin E-Cdk2 activity is dependent on the Cdk inhibitor p21 (30). Thus, the abundance and the distribution of p21 and p27 in each cell line were analyzed to determine whether these may be altered by overexpression of cyclin D1 or cyclin E. Total p21 protein peaked 15 h after treatment of the empty vector cells with ICI 182780, an effect similar to that reported in the MCF-7 cells (30). The abundance of both cyclin D1-p21 and cyclin E-p21 complexes was reduced by antiestrogen treatment in empty vector, control cells (Fig. 4,A). In contrast, both cyclin D1- and cyclin E-associated p21 was maintained in the cyclin D1-overexpressing cells (Fig. 4,B). In the cyclin E-overexpressing cells after treatment with ICI 182780, although the abundance of cyclin D1-p21 complexes decreased, the abundance of cyclin E-p21 complexes increased (Fig. 4 C).

Both p21 and p27 are critical mediators of the therapeutic effects of antiestrogen treatment (31). Whereas p21 appears to be the initiating factor in inhibition of cyclin E-Cdk2 complexes, complete inhibition of kinase activity requires the cooperation of p27 at later time points (30, 31). p27 levels increased modestly after treatment with ICI 182780 in all of the cell lines (Fig. 5). Little change in cyclin D1-p27 association was apparent in any of the cell lines (Fig. 5). However, cyclin E-p27 association increased after ICI 182780 treatment in all of the cell lines. This was apparent as early as 15 h after treatment of the cyclin E-overexpressing cell line (Fig. 5,C). The greater increase in p21 and p27 association with cyclin E-Cdk2 complexes by 48–72 h in E 17-2 cells (Fig. 4,C; Fig. 5,C) may account for the more effective inhibition of cell proliferation by the antiestrogen in the E 17-2 cells as compared with the D1 17-1 cells (Fig. 2).

Long-term effects of antiestrogen on cell growth were investigated in a colony-forming assay where cells were treated with ICI 182780 for 3 weeks. In contrast with the attenuation of the short-term antiestrogen effect on cell proliferation in short-term cultures, there was only a very slight decrease in the final level of growth inhibition produced by ICI 182780 in cyclin D1- or cyclin E-overexpressing cells in the long-term colony-forming assays (Fig. 6). Both E 17-2 and D1 17-1 cell lines were less sensitive to ICI 182780 treatment, requiring a concentration of ICI 182780 approximately 2–2.5 times greater for the same degree of inhibition (Fig. 6,B). In addition, a few colonies remained after treatment of the D1 17-1 and E 17-2 cells with high concentrations (1–10 nm) of ICI 182780 for 3 weeks (Fig. 6 B). Therefore, we investigated whether these colonies had become resistant to ICI 182780. Growth of the residual colonies resumed in the absence of ICI 182780, confirming the cytostatic nature of antiestrogen treatment. After 3 weeks of regrowth, cells were replated at the original plating density and exposed to ICI 182780 for an additional 3 weeks. Only a few colonies were obtained, similar to the original experiment, indicating sensitivity to ICI 182780 had been maintained. Thus, the few residual colonies present after 3 weeks treatment of D1 17-1 and E 17–2 cells were not due to acquired resistance to antiestrogen.

The D1 17-1 cells and to a lesser extent E 17-2 cells were resistant to ICI 182780 in the short-term, but both cell lines became sensitive to the drug in the long-term. Western blots of lysates from cyclin D1-overexpressing cells treated with ICI 182780 over this timeframe indicated that cyclin D1 protein levels were substantially reduced by long-term antiestrogen treatment (Fig. 7,A), and there was also an accompanying reduction in cyclin E-Cdk2 kinase activity (Fig. 7,B). [³⁵S]methionine/cysteine pulse-chase analysis demonstrated that the half-life of cyclin D1 protein reduced from 32 min on day 7 of treatment with ICI 182780 to 15 min on day 10 (Fig. 7,C), suggesting that the down-regulation of the protein was attributable at least in part to increased protein degradation. Although cyclin E levels were largely unchanged 7–10 days after ICI 182780 treatment of the cyclin E-overexpressing cells, a substantial increase in the cyclin E-p27 association was present at these late time points (Fig. 7,D). To determine the role of this increase in the inhibition of proliferation, p27 levels were reduced by treatment with a phosphorothioated antisense oligonucleotide. In E 17–2 cells treated with control sense oligonucleotide, pRb was almost entirely hyperphosphorylated after 1 day of treatment with ICI 182780, but only the hypophosphorylated form was evident on day 10 of treatment (Fig. 7,E), consistent with their sensitivity to antiestrogen inhibition at this time. When the p27 protein level was inhibited with an antisense oligonucleotide after 10 days of ICI 182780 treatment, pRb was hyperphosphorylated suggesting release from growth inhibition (Fig. 7 E). These data are consistent with the hypothesis that cyclin E-p27 association played an important role in the inhibition of cell growth after the long-term treatment with antiestrogen.

Endocrine therapy is an important modality of treatment in all stages of ER-positive breast cancer, and tamoxifen has been the therapy of choice for many years. Tamoxifen has demonstrated efficacy in reducing disease recurrence, mortality rate, contralateral breast cancer, and in the prevention of breast cancer in high-risk women (32, 33). However, not all of the ER-positive breast cancers respond to tamoxifen, and nearly all of the patients whose tumors initially respond will develop cellular resistance while maintaining ER-positivity (34). Given that cyclin D1 and cyclin E are overexpressed in a substantial proportion of breast cancer (7, 8) and G₁ cyclins are downstream targets of estrogen-induced mitogenesis (35, 36, 37), the cyclins are potential markers of therapeutic responsiveness to antiestrogen. The findings in this in vitro study suggest that constitutive overexpression of cyclin D1 and cyclin E interfered with the early cell cycle effects of antiestrogen inhibition. Overexpression of cyclin D1 was associated with increased S phase entry, Cdk activity, and pRb phosphorylation after antiestrogen treatment. This is consistent with the previous finding that short-term expression of cyclin D1 under the control of a zinc-inducible metallothionein promoter in ER-positive breast cancer cell lines (T-47D and MCF-7) can overcome the inhibition of cell cycle progression mediated by antiestrogens (19, 26). Overexpression of cyclin E in T-47D cells also induced some resistance over the first 24–48 h of treatment, consistent with data from tamoxifen-treated MCF-7 cells overexpressing cyclin E (24), although overexpression of cyclin E was much less effective than overexpression of cyclin D1. Despite their increased short-term resistance, T-47D cells constitutively overexpressing cyclin D1 or cyclin E became sensitive to the long-term effects of antiestrogen treatment. This phenomenon was observed previously in MCF-7 cells overexpressing cyclin D1, but the underlying mechanisms responsible for the discrepancy between the short- and long-term effects of antiestrogen were not identified (20).

Estrogens and antiestrogens interact with ER, thereby regulating the transcription of genes that control key points in G₁ progression (18, 30, 35, 36, 37). Antiestrogen treatment leads to a decrease in cyclin D1 mRNA and protein levels, inactivation of both cyclin D1-Cdk4 and cyclin E-Cdk2 complexes, and decreased pRb phosphorylation (18, 30). Previous studies of MCF-7 cells treated with ICI 182780 suggested a model of antiestrogen action in which decreased cyclin D1 abundance is an early and critical event, leading to decreased cyclin D1-Cdk4 activity and increased availability of p21 for cyclin E-Cdk2 binding (18, 30). p21 and p27 association with cyclin E-Cdk2 results in sustained inhibition of this kinase (30, 31). Although some minor differences are apparent, data presented here using a different cell line, T-47D, are consistent with the key features of this model, i.e., the essential role of decreased cyclin D1 expression and p21/p27 association with cyclin E-Cdk2. In the cyclin D1-overexpressing cells, the initial failure of antiestrogen treatment to decrease cyclin D1 expression was accompanied by maintenance of cyclin E-Cdk2 activity and pRb phosphorylation, consistent with the antiestrogen resistance of these cells after 24-h treatment and emphasizing the central role of cyclin D1.

Although the level of cyclin D1 expression in the cyclin D1-overexpressing cell line was unaffected during the first 3 days of treatment, it was significantly reduced after more extended treatment, accompanying the longer-term sensitivity of these cells to antiestrogen treatment. The down-regulation of the expression of cyclin D1 in a constitutively overexpressing cell line was unexpected and suggests an increase in the degradation of the protein as a likely mechanism, perhaps via ubiquitin-dependent proteolysis because this is a known mechanism for cyclin D1 degradation (38, 39, 40). Data demonstrating a reduction in half-life of cyclin D1 protein after 10 days of treatment support this hypothesis of a novel mechanism of antiestrogen action apparent after sustained antiestrogen treatment. In some breast cancers, cyclin D1 overexpression is thought to result from aberrations in proteins involved in cyclin D1 degradation rather than increased mRNA abundance (39). If cyclin D1 degradation is important in long-term growth inhibition by ICI 182780 and other antiestrogens in vivo, as suggested by data presented in this manuscript, aberrations in cyclin D1 stability may have implications for response to therapy that are distinct from the consequences of other mechanisms of overexpression.

Ectopic overexpression of cyclin E shortens G₁ phase duration in fibroblasts and HeLa cells (4, 41, 42), and diminishes the serum requirement of cells (42, 43). In cells with inactivation of cyclin D1-Cdk4 by overexpression of p16, pRb can still be phosphorylated by overexpression of cyclin E, indicating that tumors can gain a growth advantage by overexpression of cyclin E (44). Moreover, the phenotypic manifestations of cyclin D1 deficiency can be rescued by cyclin E, demonstrating that cyclin E can functionally replace cyclin D1 (23). The increased abundance of cyclin E in the overexpressing cell line E 17-2 led to an increase in Cdk2 activity in the absence of antiestrogens. After antiestrogen treatment, decreased cyclin E-Cdk2 activity and pRb phosphorylation were accompanied by an increase in the association of p27 with cyclin E-Cdk2, likely resulting from decreased association of these Cdk inhibitors with cyclin D1. The modest antiestrogen resistance of the cyclin E-overexpressing cells in the short-term, the persistent increase in the association of p27 with cyclin E-Cdk2, and the apparent resumption of cell cycle progression in the presence of p27 antisense oligonucleotide suggest that the redistribution of Cdk inhibitors is a significant mechanism contributing to the sensitivity of these cells to antiestrogen.

Patients with relapsed ER-positive breast cancer after initial response to tamoxifen are often treated with second and third line endocrine therapy including aromatase inhibitors and progestin. Given that the major source of estrogen in postmenopausal women is the peripheral aromatization of estrogen and androgen precursors, the enzyme aromatase has become a major molecular target for endocrine treatment (45, 46, 47). Synthetic progestins are an effective therapy in breast cancer (48) and have been used as preferred second-line hormonal agent (49) until the recent emergence of more selective aromatase inhibitors (50). In a parallel study we have shown recently that overexpression of cyclin D1 and to a lesser extent cyclin E can confer resistance to both short- and long-term progestin treatment in T-47D breast cancer cells (51). Although resistance to progestin in cyclin D1-overexpressing breast cancers requires confirmation in the clinical setting, these in vitro data support the findings from the Phase III clinical trials indicating superiority of aromastase inhibitors over progestin (50).

Given that most tumors treated with antiestrogen or other endocrine therapy are positive for both ER and cyclin D1, it is important to address whether cyclin D1 has any effect on hormonal responsiveness in the clinical setting. Our previously published study showed that a high level of cyclin D1 mRNA was a predictor for worse prognosis with increased risk of relapse, local recurrence, metastases, and death in ER-positive breast cancer (10). Additional subgroup analysis suggested that high cyclin D1 mRNA level was associated with shorter response duration in primary tamoxifen treatment (10). This hypothesis was additionally supported by the observation that failure to express both cyclin D1 and ER was a marker of poor prognosis in breast cancer treated with tamoxifen (12).

Numerous mechanisms for the eventual failure of tamoxifen treatment in ER-positive breast cancers have been proposed including elevated estrogen levels, increased tumor antiestrogen binding sites, receptor mutations, impaired signal transduction, or alteration of estrogen response elements (34, 52, 53). An increase in estrogen levels or sensitivity may in turn induce transcriptional activation of cyclin D1 expression and potentially increase cyclin D1 protein stability. Given that ectopic overexpression of cyclin D1 can overcome the cell cycle arrest of breast cancer cells (19, 26), escape from the antiestrogen-induced down-regulation of cyclin D1 may be a potential mechanism leading to endocrine resistance after long-term tamoxifen treatment. Additional clinical studies to correlate cyclin D1 expression with sensitivity to antiestrogen may help in determining the molecular basis of hormonal resistance. Although our in vitro study failed to show that ectopic overexpression of cyclin D1 had major effects on antiestrogen-induced growth inhibition, additional research is warranted to elucidate the usefulness of measurement of cyclin D1 in selecting the most efficacious endocrine therapy and the contribution of alterations in cyclin D1 expression or stability to the development of endocrine resistance in ER-positive breast cancer.

We thank Lisa-Jane Hunter for her valuable assistance with some experimental procedures.