Contrasting Effects of Prenyltransferase Inhibitors on Estrogen ...

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Endocrinology 144(3):989 –998 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-220726

Contrasting Effects of Prenyltransferase Inhibitors on Estrogen-Dependent Cell Cycle Progression and Estrogen Receptor-Mediated Transcriptional Activity in MCF-7 Cells SOPHIE F. DOISNEAU-SIXOU, PHILIPPE CESTAC, SARAH CHOUINI, JASON S. CARROLL, ANDREW D. HAMILTON, SAID M. SEBTI, MARC POIROT, PATRICK BALAGUER, JEAN-CHARLES FAYE, ROBERT L. SUTHERLAND, AND GILLES FAVRE De´partement “Innovation The´rapeutique et Oncologie Molećulaire” (S.F.D.-S., P.C., S.C., M.P., J.-C.F., G.F.), Centre de Physiopathologie de Toulouse Purpan, Institut National de la Sante´ et de la Recherche Me´dicale U563, and Institut Claudius Regaud, 31052 Toulouse cedex, France; Cancer Research Program (S.F.D.-S., J.S.C., R.L.S.), Garvan Institute of Medical Research, St. Vincent’s Hospital, Darlinghurst, Sydney, New South Wales 2010, Australia; Department of Chemistry (A.D.H.), Yale University, New Haven, Connecticut 06511; Drug Discovery Program (S.M.S.), H. Lee Moffitt Cancer Center and Research Institute and Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida 33612; and Institut National de la Sante´ et de la Recherche Me´dicale 439 (P.B.), Pathologie Molećulaire des Rećepteurs Nucleáires, 34090 Montpellier, France steroid receptor coactivator-1 with ER␣ and FTI-277 decreases the association of ER␣ with the histone deacetylase 1, a known transcriptional repressor. In addition, FTI-277 has no marked effect on the association of the two corepressors, nuclear receptor corepressor and silencing mediator of retinoid and thyroid receptor with ER␣, whereas GGTI-298, similar to tamoxifen, clearly increased these associations. Together, these results demonstrate that prenylated proteins play a role in estradiol stimulation of proliferation and progesterone receptor expression. However, they antagonize the ability of ER␣ to stimulate estrogen response element-dependent transcriptional activity, acting presumably through coregulator complex formation. (Endocrinology 144: 989 –998, 2003)

Activation of estrogen receptors (ERs) by estrogens triggers both ER nuclear transcriptional activity and Src/Ras/Erks pathway-dependent mitogenic activity. The present study implicates prenylated proteins in both estrogenic actions. The farnesyltransferase and geranylgeranyltransferase I inhibitors (FTI-277 and GGTI-298, respectively) antagonize estradiol-stimulated cell cycle progression, progesterone receptor, cyclin D1, and c-Myc expression. In contrast, the inhibitors markedly stimulate transcription from two genes containing estrogen response elements, both in the absence and presence of estradiol. The pure antiestrogen ICI 182,780 inhibits by more than 85% these effects on transcription. We demonstrate that both FTI-277 and GGTI-298 increase the association of

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HE MAJOR MITOGEN for breast epithelial cells is 17␤estradiol (E2), but it is now well accepted that peptide growth factors play important roles in controlling both mammary gland development and breast cancer progression. Most E2 actions are mediated through its nuclear receptors, estrogen receptors (ERs) ␣ and ␤, and in cancer development, growth factors have been shown to act synergistically with E2 (1, 2). For example, epidermal growth factor (EGF) is able to activate the ER and promote transcription from an estrogen response element (ERE)-containing promoter in the absence of E2. In particular, MAPK activated by growth factors, phosphorylates ER and potentiates the activation function 1 transactivation function of ER (3, 4). It has also been shown that activation of ER by E2 leads to two cascades of events that may be independent: 1) nuclear transcriptional activity

of ERs and 2) activation of Src/Ras/Erk pathway that allows S-phase entry of cells (5). Phosphatidylinositol-3 kinase inhibitors as well as inhibitors of MAPK-1 have been shown to decrease the numbers of cells entering DNA synthesis after treatment with E2 (6), but this issue remains controversial because it is not clear which pathway supports the mitogenic action of E2 (7). All these data highlight evidence for crosstalk between E2 and growth factor-induced cytoplasmic signaling. Major components of these signaling pathways are low-molecular-weight GTPases such as Ras that require prenylation for function. Ras mediates EGF phosphorylation of ER (3, 4) as well as E2 mitogenic activity in human mammary adenocarcinoma cells lines (5). Moreover, enhanced transcriptional activity of ER contributes to oncogenic K-Ras-mediated NIH-3T3 cell transformation whereas 12Val-K-Ras expression increases ER-mediated transcriptional activity (8). ER has been shown to directly interact with DNA sequences in the c-Ha-ras gene, and E2 is able to stimulate c-Ha-ras transcription. These data suggest that estrogen-mediated stimulation of c-Ha-ras transcription may play an important role in progression of breast cancer (9). Ras belongs to the Ras superfamily of low-

Abbreviations: DMSO, Dimethylsulfoxide; DTT, dithiothreitol; E2, 17␤-estradiol; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; FCS, fetal calf serum; FTase, farnesyl transferase; FTI, FTase inhibitor; GGTI, geranylgeranyltransferase I inhibitor; HAT, histone acetyltransferase; HDAC, histone deacetylase; N-CoR, nuclear receptor corepressor; PR, progesterone receptor; RNase, ribonuclease; SMRT, silencing mediator of retinoid and thyroid receptor; SRC, steroid receptor coactivator.

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Endocrinology, March 2003, 144(3):989 –998

molecular-weight proteins whose activity is controlled by a GDP/GTP cycle. Members of the Ras superfamily include Ras, Rho, and Rab subfamilies. The Ras and Rho proteins are posttranslationally modified by the isoprenoid lipids, farnesylpyrophosphate, and geranylgeranylpyrophosphate. Farnesyltransferase (FTase) and geranylgeranyltransferase I catalyze the covalent attachment of the farnesyl (C15) and the geranylgeranyl (C20) groups, respectively, to the carboxylterminal cysteine of prenylated proteins. Prenylation appears to be essential not only for membrane association but also for biological activity (10, 11). Several lines of evidence suggest cross-talk between E2 action and prenylated proteins. For example, it has been reported that E2 can overcome a G1 block in MCF-7 cells, induced by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (12), which inhibit protein prenylation by lowering the cellular pool of farnesylpyrophosphate and geranylgeranylpyrophosphate. The activated mutant forms of Rho family GTPases (RhoA, Rac1, and cdc42) inhibit ER transcriptional activation in MCF-7 and Ishikawa human uterine cancer cells (13). In breast cancers, Ras mutation has been observed in only 5% of cases (14); however, overexpression of Ras protein is common and has been associated with a more aggressive type of breast cancer (15). A marked increase of RhoB mRNA was observed in breast cancer cell lines and inversely correlated to the amount of EGF receptor (16). Furthermore, endogenous hyperactive Rac 3 was present in highly proliferative human breast cancer cells lines and tumor tissues (17). The Rho family GTPases (Rac, Cdc42, and in particular RhoA) are also overexpressed in breast cancers, compared with corresponding normal tissue (18). Finally, RhoC is overexpressed in inflammatory breast cancer (19, 20) and RhoC expression is now proposed as a potential marker for small breast carcinoma with metastatic potential (21). In this report, we assessed the specific role of prenylated proteins in estrogenic stimulation of MCF-7 cells. Our data implicate both farnesylated and geranylgeranylated proteins in E2 action because prenylation inhibitors inhibit cell cycle progression driven by E2 and stimulate the transcriptional activity mediated by ER␣. The current model of ER action suggests that ER modulates the rate of transcription through interactions with the basal transcription machinery and alterations in the recruitment of coactivators that modify chromatin organization at the promoter of target genes (22–26). We propose that the transcriptional stimulation of ER by the prenylation inhibitors is due to a similar shift in transcription coregulator association with ER␣. Our data demonstrate that both FTase inhibitors (FTI)-277 and geranylgeranyltransferase I inhibitors and (GGTI)-298 increase the association of the coactivator SRC-1 with ER␣ and that FTI-277 decreases the association of histone deacetylase 1 (HDAC1), which is essential for transcriptional repression, to the ER␣. Materials and Methods Materials Materials for cell culture and TRIzol reagent came from Life Technology (Cergy Pontoise, France). FTI-277 and GGTI-298 were a generous gift from S. Sebti (University of South Florida, Tampa, FL) or purchased from Calbiochem (Croydon, Victoria, Australia). Tamoxifen, ribonucle-

Doisneau-Sixou et al. • Prenylation and Estrogen Action in MCF-7 Cells

ase (RNase) A, Tween 20, and propidium iodide were purchased from Sigma-Aldrich (St. Quentin Fallavier, France; and Castle Hill, New South Wales, Australia). ICI 182,780 was provided by Dr. Alan Wakeling (AstraZeneca Pharmaceuticals, Macclesfield, UK), and RU38,486 was kindly provided by P. Van de Velde (Roussel Uclaf, Romainville, France).

Cell culture The human breast adenocarcinoma cell line MCF-7 was obtained from the American Tissue Culture Collection (Manassas, VA). The development of stable transfectants of MCF-7 cells (called MELN cells) has been described previously (27). These cells were established by transfecting MCF-7 cells with the ERE-␤-globin-tk-luc-SV-Neo plasmid and thus express luciferase in an estrogen-dependent manner. MCF-7 cells were grown routinely in RPMI 1640 and MELN in DMEM growth media, supplemented with 5% fetal calf serum (FCS) (Life Technologies, Inc.). Cells were incubated at 37 C in a humidified 5% CO2 incubator. For the experiments presented in Figs. 1–7, cells were grown for 5 d in phenol red-free medium, containing 5% dextran-coated charcoaltreated fetal calf serum. Medium was changed after 2 d. At d 5, cells were treated or not with FTase inhibitors and received estradiol (5 ⫻ 10⫺8 m) and/or inhibitors 24 h later. Both peptidomimetics FTI-277 and GGTI298 were dissolved in a solution of 10 mm dithiothreitol (DTT) in dimethylsulfoxide (DMSO) to avoid the formation of disulfide bonds in solution, as described previously (28). Tamoxifen and ICI 182,780 were dissolved in ethanol.

Cell cycle analysis For each condition, 3 ⫻ 105 cells were seeded per dish in two 100-mm diameter dishes and treated, as described above, in a final volume of 5 ml. Following treatment, the culture medium was aspirated and the cells were collected by trypsinization, washed twice with ice-cold PBS (100 ⫻ g, 5 min), and resuspended in 500 ␮l ice-cold PBS. Cells were then fixed in 1.5 ml ice-cold absolute ethanol for 30 min at 4 C, washed twice in PBS and then stained with propidium iodide (100 ␮g/ml RNase A in PBS, 0.25% Tween 20, and 50 ␮g/ml propidium iodide) for 1 h at 37 C. DNA content was determined by flow cytometry (FACScan flow cytometry system, Becton Dickinson and Co., Meglan, France), with a 488-nm laser excitation and 530-nm emission filter. The proportion of cells in G0/G1 phase of the cell cycle was calculated from the DNA histograms using the CellFit software (Becton Dickinson and Co.). Data were obtained from 104 viable cells.

Estrogen and progesterone receptor expression For each condition, 9 ⫻ 105 cells were seeded in 140-mm diameter dishes and treated, as described above, in a final volume of 15 ml. Cells were incubated for 48 h with estradiol and/or inhibitors. Quantitation of ER␣ and progesterone receptor (PR) protein levels was performed on the cytosol fraction of cells. Briefly, following treatment, the culture medium was removed, the cells were washed twice with PBS and scrapped in 350 ␮l homogenization buffer (10 mm Tris, pH 7.4, buffer containing 20 mm molybdic acid and 12 mm monothioglycerol). Cells were lysed by three cycles of freezing/thawing (⫺170 C/20 C) and then centrifuged at 100,000 ⫻ g for 60 min at 4 C. We used ABBOT ER-EIA monoclonal and ABBOT PgR-EIA monoclonal kits, according to the manufacturer’s instructions (Abbott, Rungis, France). This methodology provides an accurate estimate of the total cellular ER or PR levels (29). Cytosolic protein concentrations were measured using the Bradford technique (30). For each condition, average receptor concentration was calculated from the data of two independent dishes.

pS2 expression analysis The quantitation of pS2 protein was performed from the culture medium of cells growing in 140-mm-diameter dishes. We used ELSApS2 radioimmunometric assay, according to the manufacturer’s instructions (CIS Biointernational, Oris Group, Gif-sur-Yvette, France). For each condition, average pS2 concentration was calculated from the data of two independent dishes.


RNase protection assay for pS2, ER, and PR mRNA expression analysis For each condition, 9 ⫻ 105 cells were seeded per dish in three to five 140-mm dishes and treated, as described above, in a final volume of 15 ml. Cells were incubated for 48 h with estradiol and/or inhibitors. At the end of the incubation, total RNA was isolated according to the method of Chomczynski and Sacchi (31) using TRIzol reagent. Four probes were used for the RNase protection assay experiments: PR, ER, pS2, and 36B4 (32). They were the products of an in vitro transcription in the presence of [33P]UTP (3000 Ci/mmol, ICN) of the corresponding cDNA fragments cloned in a plasmid containing the T3 and T7 (for PR, ER, and 36B4) or T7 and SP6 (for pS2) promotors, in opposite directions. For mRNA quantitation, the probe used for PR was a 460-bp fragment of the complete cDNA, for pS2 a 300-bp fragment, for ER a 379-bp fragment, and for 36B4 a 650-bp fragment, all provided by Pr. Chambon (Institut de Ge´ ne´ tique et de Biologie Mole´ culaire et Cellulaire-Centre National de la Recherche Scientifique, Strasbourg, France). Fifty micrograms total RNA were incubated in the presence of pS2 and 36B4 probes (or 80 ␮g total RNA in the presence of PR or ER probes), and 5 ⫻ 105 to 106 cpm of each probe were used in hybridization buffer; samples were then treated according to RPA II kit instructions (Ambion, Inc., Austin, TX). Following digestion of nonhybridized material, hybridized RNA was purified, resuspended in electrophoresis sample buffer, and heated to 95 C before analysis on a denaturing polyacrylamide gel (8 m urea, 5% acrylamide). After fixation in a 10% acetic acid/10% methanol solution, the gel was dried and analyzed using a Storm 840 (Amersham Pharmacia Biotech, Orsay, France). The intensity of the band corresponding to the respective mRNA was quantified using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

Western blot analysis At the completion of the experiments, MCF-7 cell monolayers were washed with ice-cold PBS (Sigma-Aldrich) and the cells from two 25-cm2 flasks were then scraped into 75 ␮l ice-cold lysis buffer: 50 mm HEPES, pH 7.5; 150 mm NaCl; 10% (vol/vol) glycerol; 1% Triton X-100; 1.5 mm MgCl2; 1 mm EGTA; 100 mm NaF; 10 mm pyrophosphate; 10 ␮g/ml aprotinin; 10 mg/ml leupeptin; 1 mm phenylmethylsulfonyl fluoride; 200 ␮m sodium orthovanadate; 1 mm DTT; and 20 ␮m MG132. The lysates were then placed on ice, vortexed vigorously at intervals for 10 min, centrifuged at 15,000 ⫻ g for 10 min at 4 C, and the supernatants stored at ⫺80 C. Equal amounts of total protein (5–30 ␮g) were separated by SDS-PAGE and then transferred to polyvinyl difluoride membranes. Proteins were visualized using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) after incubation (overnight at 4 C for primary and 1 h at room temperature for secondary antibodies) using the following primary antibodies: c-Myc (SC-40) and HDAC1 (SC-7872) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); cyclin D1 (DCS-6) from Novocastra Laboratories Ltd (Newcastleupon-Tyne, UK); SRC-1 (SRC1 Ab-1) from Neomarkers (Fremont, CA); ␤-actin (AC-15) from Sigma-Aldrich, and secondary antibody antimouse horseradish peroxidase and antirabbit horseradish peroxidase from Santa Cruz Biotechnology, Inc. Nuclear receptor corepressor (N-CoR) and SMRT rabbit antisera were kindly provided by Dr. M. A. Lazar (University of Pennsylvania, Philadelphia, PA). Protein abundance was quantitated by analysis of autoradiographs. Relative band intensities were quantified by densitometric analysis (Molecular Dynamics, Inc.). Quantitation of protein levels by this method was linear over the analyzed range of protein concentrations and exposure times employed in these studies.

Luciferase assays For each condition, 15 ⫻ 103 cells were seeded per well in 12-well plates and treated, as described above, for 16 h in a final volume of 0.5 ml. At the end of the treatment, cells were washed with PBS and lysed in 150 ␮l lysis buffer (Promega Corp., Charbonnie`res, France). Luciferase activity was measured using the luciferase assay reagent (Promega Corp.), according to the manufacturer’s instructions. Protein concentrations were measured using the Bradford technique (30) to normalize the luciferase activity data. For each condition, average luciferase activity was calculated from the data of three independent wells.

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ER␣-binding studies Binding studies were performed as described earlier (33): ER was extracted from COS-7 cells transfected with pSG5-HG0 using the Lipofectamine methodology according to the manufacturer’s instructions (Life Technologies, Inc.). Briefly, transfected COS-7 cells were grown to 80% confluency in DMEM supplemented with 10% FCS, scraped, and washed twice with PBS. After centrifugation for 10 min at 120 ⫻ g, the cells were resuspended in TM buffer (20 mm Tris-HCl, pH 7.4; 20 mm sodium molybdate). Cells were broken by freeze-thaw lysis of the cell pellets in an equal volume of TM buffer. Cytosols were prepared by a 105,000 ⫻ g centrifugation at 0 C for 60 min. Glycerol was added to the cytosol to a final concentration of 10% (vol/vol), the extract frozen immediately in liquid nitrogen, and stored at ⫺80 C until use. The cytosol was subsequently diluted to 1:10 in TM buffer and then incubated for 18 h at 4 C in a volume of 100 ␮l with 10 ␮g protein and 2 nm [3H] estradiol (91 Ci/mmol, Amersham Pharmacia Biotech), with or without 25 ␮m unlabeled ligand. Assays were terminated by loading 65 ␮l incubate on a 1.2-ml Sephadex LH-20 (Sigma-Aldrich) column equilibrated with the TM buffer. The flow-through was collected and counted for radioactivity in ready Emulsifier-safe scintillant (Packard Bioscience B.V., Gronningen, The Netherlands).

Detection of ER␣-associated proteins The 150-cm2 flasks were seeded with 1 ⫻ 106 cells. Cells were allowed to proliferate for 2 d in normal RPMI 1640 medium supplemented with 5% nontreated FCS until they reached 50% confluence, after which drugs or vehicle were added directly to the medium. Cells were treated with FTI-277, GGTI-298, tamoxifen, or vehicle. The final concentrations of ethanol and DTT/DMSO solution in the medium were 0.5% and had no effect on the rate of cell proliferation (data not shown). After 48 h, MCF-7 cell monolayers were washed with ice-cold PBS (Sigma), and the cells from three 150-cm2 flasks were then scraped into 600 ␮l ice-cold lysis buffer (as described above for Western blot analysis). The lysates were then placed on ice, vortexed vigorously at intervals for 10 min, centrifuged at 15,000 ⫻ g for 10 min at 4 C, and the supernatants stored at ⫺80 C. Lysates were obtained and stored as described above. Coregulator complexes were immunoprecipitated from equivalent amounts of protein (500 ␮g) with goat polyclonal antihuman N-CoR (SC-1609) or SMRT (SC-1610) antibodies or rabbit polyclonal antihuman HDAC1 (SC-7872) or SRC-1 (SC-8995) antibodies (Santa Cruz Biotechnology, Inc.), for 1 h at 4 C. This was followed by a 1-h incubation at 4 C with protein A-Sepharose or protein G-Sepharose (Zymed Laboratories, Inc. Corp., San Francisco, CA) for conjugation to rabbit and goat antibodies, respectively. Immunoprecipitates were washed twice with ice-cold lysis buffer containing 1 m NaCl and then twice with ice-cold 50 mm HEPES, pH 7.5; 1 mm DTT. Proteins were separated from protein A- or protein G-Sepharose beads by resuspending the beads in 1⫻ SDS sample buffer and heating at 95 C for 3 min. Proteins were then separated by SDS-PAGE and transferred to polyvinyl difluoride membrane. They were visualized using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) after overnight incubation at 4 C using the mouse antihuman ER␣ antibody (M7047, DAKO Corp.). Protein abundance was visualized by densitometric analysis (Molecular Dynamics, Inc.).

Statistical analysis Statistical analysis of the data were conducted using an unpaired two-sample t test. Significance was defined as P ⬍ 0.05.

Results Geranylgeranylated and farnesylated proteins are required for E2-stimulated MCF-7 cell cycle progression

We analyzed Ras and Rap1A prenylation (proteins, respectively, farnesylated and geranylgeranylated) as described previously (28) and determined the maximal concentrations of inhibitors that efficiently and specifically inhibit protein prenylation in MCF-7 cells after 48-h incubation. We found that 15 ␮m FTI-277 and 10 ␮m GGTI-298

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potently and selectively inhibited H-Ras farnesylation and Rap geranylgeranylation, respectively (data not shown). To determine whether prenylated proteins are implicated in E2 stimulation of MCF-7 cells, we first assessed the effect of FTI-277 and GGTI-298 on cell cycle progression after E2 stimulation (Fig. 1A). After 5 d of E2 deprivation, cells were stimulated with E2 in the presence or absence of inhibitors. In the absence of inhibitors, E2 stimulated S-phase entry and hence significantly decreased the proportion of cells in G0/G1 phase of the cell cycle. In the presence of FTI-277, fewer cells entered S phase with a higher percentage of cells remaining in G0/G1 phase. In the presence of GGTI-298, the block in G0/G1 phase was almost complete. Thus, GGTI-298 and, to a lesser degree, FTI-277 prevented E2-stimulated cell cycle progression, suggesting that both farnesylation and geranylgeranylation of proteins are required for E2-stimulated cell cycle progression in MCF-7 cells. Preincubation with FTI-277, but not GGTI-298, is necessary to inhibit E2-stimulated cell cycle progression

Most prenylated proteins have a long half-life (24 h for Ras and RhoA); however, others have half-lives as short as 2 h, i.e. RhoB (34). Prenylation is a covalent posttranslational modification of proteins and prenylation inhibitors act by inhibiting prenylation of newly synthesized proteins. We

FIG. 1. Effects of prenyltransferase inhibitors on E2-stimulated MCF-7 cell cycle progression. A, Cells, deprived of E2 for 5 d, were treated with 50 nM E2 or ethanol with or without FTI-277 and GGTI298 (15 ␮M and 10 ␮M, respectively, or DTT/DMSO vehicle). The percentage of cells in G0/G1 phase of the cell cycle was analyzed 48 h after E2 addition by flow cytometry, as described in Materials and Methods. Each histogram is the mean value of 104 viable cells, and results are representative of at least three independent experiments. B, Importance of inhibitor pretreatment for their effects on cell cycle progression. Cells were deprived of E2 for 6 d, and then all cells were stimulated with E2 (50 nM). Simultaneously, cells were treated or not with FTI-277 or GGTI-298 (15 ␮M and 10 ␮M, respectively, or DTT/ DMSO vehicle). Fifty percent of cells were pretreated for 24 h with FTI-277 and GGTI-298 before E2 addition (same concentrations as above). The percentage of cells in G0/G1 phase of the cell cycle was analyzed 30 h after E2 addition by flow cytometry. Each histogram is the mean value of 104 viable cells.


therefore determined the importance of the length of exposure to prenylation inhibitors by administering the prenyltransferase inhibitors for 24 h before E2 exposure (Fig. 1B). After 6 d of E2 deprivation, cells were stimulated by E2 in the presence or absence of inhibitors and cell cycle progression was analyzed 30 h later. As expected, under control conditions, E2 allowed deprived cells to enter S phase and only about 20% of cells remained in G0/G1 phase. FTI-277 had to be added 24 h before E2 to block S-phase entry with 60% of cells remaining in G0/G1. By contrast, GGTI-298 strongly inhibited S-phase entry, even without preincubation. The dramatic effect of GGTI-298 was observed as early as 16 h after E2 addition when cells began to enter S phase (data not shown). GGTI-298 added up to 6 h after E2 inhibits E2-stimulated cell cycle progression

The observation that the simultaneous addition of E2 and GGTI-298 completely blocked S-phase entry suggested that the GGTI-298 effect occurred during early or late G1 phase. We then determined how long after E2 addition GGTI-298 efficiently inhibits cell cycle progression (Fig. 2). After 5 d of E2 deprivation, cells were stimulated with E2. GGTI-298 was added simultaneously or 3, 6, 9, 12, 15, or 18 h later and the percentage of cells in G0/G1 analyzed 30 h after E2 addition. In the absence of GGTI-298, E2 stimulated 50% of cells to enter S phase and 35% of cells remained in G0/G1. The addition of GGTI-298 at time 0 (time of addition of E2) induced a complete block of cells in G0/G1 (77%). The addition of GGTI-298 up to 6 h after E2 significantly impaired the E2 effect. GGTI-298 had a decreased effect if added 9 h after E2 addition and little or no effect at later times. In subsequent experiments, a 24-h incubation with FTI277, before E2 addition, was performed, whereas GGTI-298 was added simultaneously with E2.

FIG. 2. Kinetic effect of GGTI-298 addition on E2-induced MCF-7 cell cycle progression. Cells, deprived of E2 for 5 d, were stimulated with E2 (50 nM) at time 0 and treated or not with GGTI-298 (10 ␮M). Addition of GGTI-298 was performed at time 0, 3, 6, 9, 12, 15, or 18 h after E2 addition. The percentage of cells in G0/G1 phase was analyzed 30 h after E2 addition by flow cytometry. Each data point is the mean value of 104 viable cells, and results are representative of three independent experiments.



Contrasting roles of prenylated proteins in E2-stimulated protein expression

The pS2 expression was next analyzed under identical conditions (Fig. 4). Figure 4A shows that E2 alone increased pS2 protein levels by 6.7-fold. In the presence of FTI-277, the induction was about the same, whereas GGTI-298 allowed an 18-fold induction. Without E2, but in the presence of FTI-277, there was no change of the basal pS2 protein level, whereas in the presence of GGTI-298, a 1.9-fold induction was observed. In Fig. 4B, a 2.6-fold induction of pS2 mRNA level was observed after E2 addition, a nonsignificantly different 3.3-fold induction in the presence of FTI-277, and a statistically significant 6.8-fold induction in the presence of GGTI298. Without E2 and in the presence of FTI-277, there was no change of the basal level, whereas in the presence of GGTI298, a 1.7-fold induction was observed.

We next analyzed the effect of prenyltransferase inhibitors on the expression of two well-known ER transcriptional targets, PR and pS2. We first assessed the effect of FTI-277 and GGTI-298 on PR expression after E2 stimulation (Fig. 3). After 5 d of E2 deprivation, cells were treated with E2 in the presence or absence of inhibitors, and PR protein concentrations (Fig. 3A) and mRNA levels (Fig. 3B) were determined 48 h later. E2 alone induced PR protein levels by 5.8-fold (Fig. 3A). In the presence of FTI-277 or GGTI-298, the induction was decreased to 3.6-fold and 1.9-fold, respectively. The effects of both inhibitors were statistically different from the E2-stimulated control (t test). In the absence of E2, FTI-277 had no effect on basal PR protein levels, whereas GGTI-298 decreased PR protein levels by 58%. A 6.3-fold induction of PR mRNA level was observed after E2 addition (Fig. 3B), and in the presence of FTI-277 or GGTI-298, induction was decreased to 4.8-fold and 4.3-fold, respectively, but this was not statistically significant. Thus, GGTI-298 and, to a lesser degree, FTI-277 significantly inhibited E2-stimulated PR expression and the same trend was observed at the mRNA level.

FIG. 3. Effect of prenyltransferase inhibitors on PR expression in MCF-7 cells. Cells, deprived of E2 for 5 d, were treated or not with FTI-277 (15 ␮M or DTT/DMSO vehicle). Twenty-four hours later, they were stimulated with E2 (50 nM) or ethanol and treated or not with FTI-277 and GGTI-298 (respectively, 15 ␮M and 10 ␮M, or DTT/DMSO vehicle). Analyses were performed 48 h after E2 addition, as described in Materials and Methods. Results are expressed as a percentage of non-E2-stimulated PR protein (A) and mRNA (B) levels. In A, 100% is equivalent to 58.2 fmol PR per milligram of protein. In B, the arrow indicates the position of the band corresponding to the PR probe for RNase mapping. Error bars indicate the mean values ⫾ SEM from four independent experiments; they are plotted for all experimental conditions but are not visible on some histograms when too small to be distinguished from the mean.

Prenylated proteins positively modulate expression of early response genes

Because both E2 and the prenyltransferases inhibitors have multiple effects after 48 h, we determined the effects of the inhibitors at earlier time points by analyzing their effect on expression of two early response genes involved in the mitogenic response to E2: c-Myc and cyclin D1. After 5 d of E2 deprivation, cells were treated with inhibitors for 24 h before

FIG. 4. Effect of prenyltransferase inhibitors on pS2 expression in MCF-7 cells. Cells, deprived of E2 for 5 d, were treated or not with FTI-277 (15 ␮M or DTT/DMSO vehicle). Twenty-four hours later, they were stimulated with E2 (50 nM) or ethanol and treated or not with FTI-277 and GGTI-298 (respectively,15 ␮M and 10 ␮M, or DTT/DMSO vehicle). Analyses were performed 48 h after E2 addition, as described in Materials and Methods. Results are expressed as a percentage of non-E2-stimulated pS2 protein level in the culture medium (A) and pS2 mRNA (B). In A, 100% is equivalent to 6.55 ng pS2 protein per milliliter of medium. In B, the arrow indicates the position of the bands corresponding to pS2 and 36B4 probes for RNase mapping. Error bars indicate the mean values ⫾ SEM from three independent experiments; they are plotted for all conditions but are not visible on some histograms when too small to be distinguished from the mean.

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E2 was added. The c-Myc (Fig. 5A) and cyclin D1 (Fig. 5B) expression was determined 3 and 6 h later. For both c-Myc and cyclin D1, E2 alone induced the protein levels by 3- and 1.5-fold, respectively, within 3 h of E2 addition. Although FTI-277 and GGTI-298 already inhibited c-Myc at 3 h, they both tended to decrease the expression at 6 h of c-Myc (by 13% and 30%, respectively) and cyclin D1 (by 30%). Prenylated proteins positively modulate ER expression

To determine whether some of the effects of prenyltransferase inhibitors (such as the decrease of PR expression or induction of pS2 expression) can be explained by changes in ER␣ expression, ER␣ levels were quantified under the conditions previously described (Fig. 6). Figure 6A shows that in the absence of E2, FTI-277, and GGTI-298 decreased ER␣ protein levels by 66% and 76%, respectively. Figure 6A also shows that, as expected, E2 alone induced a 66% decrease in the ER␣ protein levels. In the presence of E2 and FTI-277 or GGTI-298, a further decrease was observed with GGTI-298 but not with FTI-277. In Fig. 6B, a marked decrease in the basal level of ER mRNA was observed with E2, FTI-277, or GGTI-298. Similar to the effect on protein levels, FTI-277 or GGTI-298 alone decreased the basal level of ER mRNA in the absence of E2. FTIs and GGTI-298 markedly enhance ER-mediated transcription in MELN cells

Because we demonstrated that prenylation inhibitors are able to stimulate pS2 expression and inhibit PR expression, we next determined whether they act directly on ER transcriptional activity. For this, we analyzed their effects on an

FIG. 5. Effect of prenyltransferase inhibitors on c-Myc and cyclin D1 expression in MCF-7 cells. Cells, deprived of E2 for 5 d, were treated or not with FTI-277 and GGTI-298 (respectively, 15 ␮M and 10 ␮M, or DTT/DMSO vehicle). Twenty-four hours later, they were stimulated with E2 (50 nM) or ethanol and treated or not with FTI-277 and GGTI-298 (respectively, 15 ␮M and 10 ␮M, or DTT/DMSO vehicle). Analyses were performed 3 and 6 h after E2 addition, as described in Materials and Methods. Results are expressed as a percentage of non-E2-stimulated c-Myc (A) and cyclin D1 (B) protein levels. These experiments are representative of two independent experiments.

FIG. 6. Effect of prenyltransferase inhibitors on ER expression in MCF-7 cells. Cells, deprived of E2 for 5 d, were treated or not with FTI-277 (15 ␮M or DTT/DMSO vehicle). Twenty-four hours later, they were stimulated with E2 (50 nM) or ethanol and treated or not with FTI-277 and GGTI-298 (respectively, 15 ␮M and 10 ␮M, or DTT/DMSO vehicle). Analyses were performed 48 h after E2 addition, as described in Materials and Methods. Results are expressed as a percentage of non-E2-stimulated ER protein (A) and mRNA (B). In A, 100% is equivalent to 199.3 fmol of ER per milligram of protein. In B, the arrow indicates the position of the band corresponding to ER probe for RNase mapping. Error bars indicate the mean values ⫾ SEM from three independent experiments; they are plotted for all conditions but are not visible on some histograms when too small to be distinguished from the mean.

ERE-dependent luciferase reporter gene, stably expressed in MCF-7 cells (MELN cells) (27). After 5 d of E2 deprivation, cells were treated with E2 in the presence or absence of prenyltransferase inhibitors or ICI 182,780 (Fig. 7), and 16 h later, luciferase activity was quantified. In vehicle-treated cells, a 10.4-fold induction of the luciferase activity by E2 was observed. This induction was inhibited by 80% in the presence of ICI 182,780. In the absence of E2, FTI-277 and GGTI-298 stimulated basal transcriptional level by 5- and 3-fold, respectively. The results shown in Fig. 7 are representative of nine and six independent experiments, respectively, with values of 5 ⫾ 3.3-fold for FTI-277 and 3.7 ⫾ 1.8-fold for GGTI-298. In the presence of E2, FTI277 and GGTI-298 enhanced the ability of E2 to stimulate transcription by an additional 2.8 ⫾ 1.3-fold for FTI-277 and 2.9 ⫾ 1.3-fold for GGTI-298 –277 (10 and 6 experiments, respectively). In both cases, ICI 182,780 inhibited the activity by at least 80%. We then determined the effect of the RU38,486, an antagonist of PR and the glucocorticoid receptor, and showed that it had no significant effect on luciferase activity in the absence or presence of FTI-277 and GGTI-298 (data not shown).



FIG. 7. Effect of prenyltransferase inhibitors on ERE-dependent luciferase activity in MELN cells in the presence or absence of ICI 182,780. Cells, deprived of E2 for 5 d, were treated or not with FTI-277 (15 ␮M or DTT/DMSO vehicle). Twenty-four hours later, they were stimulated with E2 (50 nM) or ethanol and treated or not with FTI-277 and GGTI-298 (respectively, 15 ␮M and 10 ␮M, or DTT/DMSO vehicle) and ICI 182,780 (500 nM). Luciferase activities were quantified 16 h after E2 addition, as described in Materials and Methods. Results are expressed in arbitrary units after protein normalization. Error bars indicate the mean values ⫾ SD from triplicate experiments, and results are representative of two independent experiments.

To determine whether the effects we observed are related to the specific structure of FTI-277 and GGTI-298 (peptidomimetics), we tested under identical conditions the effects of two other FTIs of different chemical structures and demonstrated that they all increased luciferase activity in the absence as well as presence of E2 (data not shown). In all cases, the stimulation was strongly inhibited by ICI 182,780. We also demonstrated that these inhibitors do not compete directly with E2 binding. For this, competition analysis of [3H]estradiol binding to ER␣ was performed in the presence of FTI-277, GGTI-298, and two other FTIs of different structures. None of these compounds exhibited any displacement of [3H]estradiol, demonstrating their lack of binding to ER␣ (data not shown). FTI-277 and GGTI-298 modulate the association of coregulators with ER␣

It has been shown that in mammary cells, tamoxifen promotes the binding of ERs to the nuclear receptor corepressor N-CoR and the related factor, silencing mediator of retinoid and thyroid receptors (SMRT), and that the relative expression levels of coactivators and corepressors may modulate the ability of tamoxifen to regulate ER transcriptional activity (22–25). More recently cell type- and promoter-specific differences in coregulator recruitment have been shown to determine the cellular response to selective estrogen receptor modulators, such as tamoxifen (26). We therefore assessed the ability of tamoxifen, FTI-277, and GGTI-298 to regulate ER␣ association with various coregulators in the presence of E2 (Fig. 8A). As expected (26), tamoxifen increased the association of N-CoR and SMRT with ER␣ and dramatically enhanced HDAC1 association. FTI-277 slightly increased NCoR association with ER␣ but was less potent than tamoxifen and had no effect on SMRT association with ER␣. In addition, FTI-277 induced the complete dissociation of HDAC1 from

FIG. 8. Effect of tamoxifen, FTI-277, and GGTI-298 on various coregulators of transcription. MCF-7 cells were grown for 48 h in RPMI 1640 growth medium, supplemented with 5% FCS. Cells were then treated with tamoxifen (1 ␮M), FTI-277 (15 ␮M), GGTI-298 (10 ␮M), or vehicle. Whole-cell lysates were prepared at 48 h. A, Effect of the inhibitors on ER␣ association to the coregulators of transcription. Each of the four coregulators was immunoprecipitated from 500 ␮g of the same lysates. The immunoprecipitates were subjected to electrophoresis on 10% SDS-PAGE and immunoblotted with anti-ER␣ antibody. B, Effect of tamoxifen, FTI-277, and GGTI-298 on coregulator expression levels. Thirty micrograms total protein was separated by 6% and 10% SDS-PAGE and immunoblotted with coregulator and ␤-actin antibodies or sera. Total protein levels were visualized using densitometry as described under Materials and Methods. The results are representative of two independent experiments.

ER␣. Although GGTI-298 did not impair the association of HDAC1 with ER␣, it unexpectedly increased ER␣ association with N-CoR and SMRT, even more so than tamoxifen. These effects observed in the presence of GGTI-298 are particularly marked, considering the fact that under these experimental conditions, ER␣ levels are about one third of those seen under the control conditions (Fig. 6). FTI-277 and GGTI-298 also significantly increased the association of the coactivator SRC-1 with ER␣ when compared with the control or tamoxifen-treated cells in which the association was considerably weaker. A Western blot analysis of SMRT, N-CoR, SRC-1, and HDAC1 was performed to determine whether the inhibitors change their expression levels, potentially explaining the altered levels of association with ER␣. The data shown in Fig. 8B indicate that the inhibitors and tamoxifen have no effect on SRC-1, whereas tamoxifen, FTI-277, and to a lesser extent GGTI-298 decrease SMRT and N-CoR levels. Regarding the HDAC1 levels, we observed a decrease in the presence of

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both tamoxifen and FTI-277 although the signals were very weak (data not shown). Consequently, none of these changes explain the alterations in ER␣ association. Discussion

The classical model of E2 action involves binding to nuclear receptors ER␣ and ER␤, which interact with chromatin to modulate the activity of target gene promoters. Besides this genomic pathway, E2 induces nongenomic effects (often immediate and transient) via signaling pathways that may involve plasma membrane or cytoplasmic ER (1, 5, 35–37). Several mechanisms have been proposed to account for the mitogenic action of E2, including 1) hormone-regulated autocrine pathways involving polypeptide growth factors and 2) a direct mitogenic action mediated either by membranebound receptors (38 – 40) or activation of hormone-independent pathways involving tyrosine kinases or second messengers or the nuclear ER directly activating the expression of genes known to be rate limiting to cell cycle progression, e.g. c-Myc and cyclin D1 (12, 41– 43). However, the exact mechanisms that mediate the mitogenic actions of E2 action are yet to be fully defined. Because many prenylated proteins are components of signal transduction pathways stimulated by E2, we studied the effects of the prenyltransferase inhibitors, FTI-277 and GTI298, on E2-stimulated cell cycle progression in MCF-7 cells. The inhibitors led, respectively, to partial and complete inhibition of cell cycle progression demonstrating that both farnesylated and geranylgeranylated proteins are involved in the mitogenic response to estrogen. We selected the established model of E2-deprived MCF-7 cells to specifically assess the role of prenylated proteins in E2-induced mitogenesis because it is known that FTIs such as the FTI L744,832 inhibit the growth of over 70% of human tumor cell lines, four of seven breast cancer cell lines and is unrelated to their ER status (44). The requirement for protein prenylation for E2-stimulated proliferation is consistent with its requirement for E2-stimulated PR expression both at the protein and mRNA levels. This is in good agreement with the therapeutic behavior, where responses to endocrine therapy are increased in ER⫹/ PR⫹ breast cancers. However, the PR gene does not possess any consensus EREs, and its expression is not integral to the proliferative response to estrogens because PR⫺/⫺ mice develop normally at puberty and respond to repeated administration of estrogens with dramatic hyperplasia of the uterus (45). Similarly, both c-Myc and cyclin D1 expression are rapidly induced by E2 and trigger cell cycle progression, although the corresponding gene promotors contain no ERElike sequence (46, 47). Our results show that prenylated proteins may be involved in the induction of c-Myc and/or maintain the expression level of these proteins after E2 stimulation because they do not interfere with the early induction of cyclin D1 at 3 h. Both FTI-277 and GGTI-298 repress ER expression (at the mRNA and protein levels) mimicking an E2-like effect. GGTI-298 dramatically increases pS2 expression in the presence and absence of E2, clearly demonstrating the involvement of geranylgeranylated proteins in the negative regu-


lation of pS2 expression, including its basal expression. The pS2 gene possesses an imperfect palindromic ERE sequence (48). In MELN cells, transcriptional activation of an EREdependent luciferase gene in the absence and presence of E2 is induced by FTI-277 and GGTI-298. Besides these peptidomimetic prenyltransferase inhibitors, we checked that FTIs of two different chemical structures have similar effects (data not shown). None of these four compounds exhibit binding to ER␣, but their stimulatory effects on ERE-dependent transcription are prevented by ICI 182,780 in the presence as well as absence of E2. These data strongly suggest that farnesylated and geranylgeranylated proteins play a role in the negative control of the ER-mediated transcriptional activity. Although there is no definitive evidence that the effects of the inhibitors are specific to ER activity, these data are consistent with the demonstration that the prenylated Rho GTPases are important modulators of ER transcriptional activity (13). The presence of an identifiable ERE may determine the role of the prenylated proteins on ERE-mediated transcription, as suggested by our results. Nevertheless, the activity of a number of transcription factors, which may determine the overall promotor activity of ERE-containing genes, is known to be altered by prenylated proteins (e.g. SRF, nuclear factor ␬B) (49 –51). The ER is a ligand-activated transcriptional scaffold that, on activation, results in conformational changes in the receptor followed by recruitment of various transcription coactivators that may be general or receptor specific, such as the histone acetyltransferase (HAT), cAMP response elementbinding protein/p300, and the steroid receptor coactivator (SRC)-1 (22–25). They form multiprotein complexes responsible for transcription initiation by remodeling chromatin through their intrinsic HAT activity (52). The transcriptional response to specific ligands depends on the ability of the ER complex to modulate the switch between coactivator complexes with HAT activity and corepressor complexes with associated histone deacetylase activity (N-CoR and SMRT) (reviewed in Ref. 52). Histone deacetylase proteins comprise a family of proteins that act in conjunction with HAT proteins to modulate chromatin structure and transcriptional activity via changes in the acetylation status of histones. Histones H3 and H4 are the principal histone targets of HDAC enzymatic activity. The association of HDAC proteins with mSin3, NCoR, SMRT, or other transcriptional repressors has led to the hypothesis that HDAC proteins participate in transcriptional corepressor activity (53). We examined N-CoR, SMRT, and HDAC1 as corepressors and the general SRC-1 to assess changes in ER␣ association following treatment with prenyltransferase inhibitors. As expected, tamoxifen increased the binding of the two corepressors and HDAC1 to ER␣, with no significant recruitment of SRC-1. In contrast, FTI-277 recruited at least SRC-1 and dissociated HDAC1 from the ER␣ complex, without dramatic changes in corepressor association. GGTI-298 clearly induced recruitment of SRC-1 to ER␣ and, surprisingly, increased the association of the two corepressors to the ER␣ complex. Unlike FTI-277, GGTI-298 does not influence the association of HDAC1 with ER␣. It then appears that coactivator recruitment to ER complexes is an essential event that allows prenyltransferase inhibitors to increase ER-related


transcriptional activity, besides their direct effects on corepressor/coactivator expression. Our results emphasize the fact that the overall balance of the relative expression levels of coactivators and corepressors and their association with ER is essential in regulating transcriptional potential following ligand-ER association. Our work provides the first evidence of contrasting effects of FTI-277 and GGTI-298 on inhibition of E2-mediated mitogenesis and ERE-dependent transcriptional activity. This is supported by the demonstration that activation of ER by E2 may lead to these two potentially separate cascades of events (5). Many prenylated proteins are possible candidates for either of these two effects (54). A geranylgeranylated protein appears to be more potent in mediating E2-induced proliferation than a farnesylated protein. It has been suggested that RhoA, a geranylgeranylated protein, regulates cell cycle progression by modulating the protein stability of the cell cycle inhibitor p27kip1 (55) or transcription regulation of specific cell cycle regulatory genes such as c-fos (49), p21waf (56), or cyclin D1 (57). The estrogen-dependent expression of cyclin D1 is essential for estrogen-induced proliferation of MCF-7 cells (58) and is an early estrogen-mediated effect on the cell cycle machinery (41). RhoA could be eliminated from the membrane fraction during G1 progression and new RhoA synthesized for G1/S progression (59). Moreover, RhoA is overexpressed in human breast cancer cells, and its expression level is significantly higher in grade III cancers when compared with grade I cancers (18). In addition, RhoB, which can be both farnesylated and geranylgeranylated in cells, has a short half-life (2 h) and is synthesized during the S phase of the cell cycle (60). It is noteworthy that ER regulates gene transcription either by binding directly to the promoter of target genes (genes containing an ERE) or indirectly through a mechanism involving other transcription factors, such as Sp1 and activator protein-1 (AP1) (for genes containing nonclassical response sites). Because it was recently shown that AP1-mediated transcription is down-regulated by RhoB, RhoB may act on E2-mediated mitogenesis through this pathway (61). With regard to ER transcriptional activity for EREcontaining genes, our data demonstrate that farnesylated and/or geranylgeranylated proteins inhibit basal as well as E2-dependent ER activities. A genetic screen in yeast identified Rho guanine nucleotide dissociation inhibitor as a positive regulator of ER transactivation and suggest that this effect is mediated by antagonizing Rho function (13). Moreover, it has been shown that Brx, a Dbl oncogene family member, which is a guanine nucleotide exchange factor of Rho proteins, contains a nuclear hormone receptor-binding region. Brx affects ER-mediated gene activation by a mechanism that is dependent on the Cdc42Hs signaling pathway (62). Besides, constitutively active forms of c-Raf and Rac synergistically enhance the cAMP response element-binding protein/p300-mediated increase of transcription in T-cell activation signals (63). Finally, a constitutively active form of Cdc-42 induces H4 hyperacetylation in chromatin (64). Whether prenylated proteins have a direct action on the ERs is not known, but our data strongly suggest an action at the level of ER corepressor/coactivator equilibrium. This is in agreement with the emerging importance of the overall


balance of coregulators of ER complexes in regulating ERrelated transcriptional activity. The exact identity of the prenylated proteins involved in the ER coregulator complex assembly and the molecular mechanisms for transcriptional repression remains to be determined. Acknowledgments We are grateful to Dr. N. Eche and A. Margras (Institut Claudius Regaud) for their help with the pS2 analysis, Dr. M. Gardiner-Garden for the statistical analysis, and Dr. E. A. Musgrove for her invaluable contribution to the work. Received July 17, 2002. Accepted November 15, 2002. Address all correspondence and requests for reprints to: Robert L. Sutherland, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, New South Wales 2010, Australia. E-mail: [email protected]. This work was supported by Grant CA-67771 from the National Cancer Institute (to S.M.S. and A.D.H.) and grants from “Groupe de Recherche de l’Institut Claudius Regaud,” Institut National de la Sante´ et de la Recherche Me´ dicale, National Health and Medical Research Council of Australia, and the Cancer Council New South Wales.

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