(Ink4a/ARF) Locus Encoded Proteins P16ink4a and P19arf Repress Cyclin D1 Transcription Through Distinct Cis Elements

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[CANCER RESEARCH 64, 4122–4130, June 15, 2004] The Inhibitor of Cyclin-Dependent Kinase 4a/Alternative Reading Frame (INK4a/ARF) Locus Encoded Proteins p16INK4a and p19ARF Repress Cyclin D1 Transcription through Distinct cis Elements

Mark D’Amico,1 Kongming Wu,1 Maofu Fu,1 Mahadev Rao,1 Chris Albanese,1 Robert G. Russell,1 Hanzhou Lian,2 David Bregman,2,3 Michael A. White,4 and Richard G. Pestell1 1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC; Departments of 2Pathology and 3Molecular Pharmacology, Albert Einstein College of Medicine, Albert Einstein Cancer Center, Bronx, New York; and 4Department of Cell Biology and Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas

ABSTRACT cycle arrest (10–12). The tumor suppressors RASSF1A and the INI1 repressor components of the SWI/SNF complex mediate cell cycle The Ink4a/Arf locus encodes two structurally unrelated tumor suppres- arrest through repression of cyclin D1 (13, 14). As a common tran- sor proteins, p16INK4a and p14ARF (murine p19ARF). Invariant inactivation scriptional target of diverse oncogenic and mitogenic signaling path- of either the p16INK4a-cyclin D/CDK-pRb pathway and/or p53-p14ARF pathway occurs in most human tumors. Cyclin D1 is frequently overex- ways through distinct DNA sequences, cyclin D1 transcription pro- pressed in breast cancer cells contributing an alternate mechanism inac- vides the basis for collaborative induction by proliferative pathways tivating the p16INK4a/pRb pathway. Targeted overexpression of cyclin D1 (15). Conversely, transcriptional repression of cyclin D1 by cytostatic -to the mammary gland is sufficient for tumorigenesis, and cyclin D1؊/؊ molecules serves to integrate in a dynamic manner cell cycle inhibi mice are resistant to Ras-induced mammary tumors. Recent studies sug- tory effectors. gest cyclin D1 and p16INK4a expression are reciprocal in human breast The complex interplay between tumor-suppressor genes and tumor- cancers. Herein, reciprocal regulation of cyclin D1 and p16INK4a was promoting genes has been simplified through the almost invariant INK4a observed in tissues of mice mutant for the Ink4a/Arf locus. p16 and inactivation of the p53 and the p16INK4a/retinoblastoma (Rb) pathway ARF INK4a p19 inhibited DNA synthesis in MCF7 cells. p16 repressed cyclin in human cancer (16, 17). Cyclin D1 overexpression provides a D1 expression and transcription. Repression of cyclin D1 by p16INK4a common mechanism for disruption of the Rb pathway in human breast occurred independently of the p16INK4a-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-bind- cancers. In other tumor types, Rb pathway inactivation may occur as ing site. p19ARF repressed cyclin D1 through a novel distal cis-element at a result of inactivation of Rb through mutation, deletion, or inactiva- ؊1137, which bound p53 in chromatin-immunoprecipitation assays. tion of Rb function through viral sequestration, phosphorylation, or Transcriptional repression of the cyclin D1 gene through distinct DNA dysregulation of components controlling Rb phosphorylation (18). sequences may contribute to the tumor suppressor function of the Ink4a/ Mutations of G1-specific cyclin-dependent kinase catalytic units Arf locus. and/or elimination of inhibitor(s) of cyclin-dependent kinase 4 (INK4) also contribute to the phosphorylation status and inactivation of Rb INTRODUCTION (16, 17). The INK4a/ARF locus is involved in both the pRb and p53 pathways encoding p16INK4a, a regulator of cdk4/6-mediated pRB The cyclin D1 gene encodes a labile regulatory subunit of a ho- phosphorylation, and p19ARF, a modulator of Mdm2-mediated deg- loenzyme that phosphorylates and inactivates the pRb retinoblastoma radation of p53 (19, 20). Mice deleted of the Ink4/Arf locus are prone tumor suppressor. Oncogenic signals including Ras, Src, Stat3, and to spontaneous tumor formation and have increased susceptibility to ␤ -catenin induce the transcription and abundance of cyclin D1 (1–4). oncogenic effects of Ras, Myc, and an activated epidermal growth Cyclin D1 is overexpressed in more than 30% of human breast factor receptor (21–23) indicating the importance of this locus in cancers and mammary gland-targeted expression of cyclin D1 is tumor suppression in vivo. sufficient for the induction of mammary tumorigenesis (5). Con- Activating signals from ␤-catenin, Src, and the Ras-Raf-MAPK versely the reduction in cyclin D1 abundance using antisense and pathway induce INK4a/ARF (24–27). Thus common oncogenic sig- immunoneutralizing antibodies has demonstrated a requirement for nals induce expression of both cyclin and cyclin inhibitors. The cyclin D1 in proliferative signaling induced by growth factors and fidelity with which INK4a/ARF, induced by oncogenic signals, trans- hormones in breast cancer epithelial cells (6, 7). Mice deleted of the duces a tumor suppressor phenotype is of fundamental importance cyclin D1 gene are resistant to Ras- and ErbB2-induced mammary (16, 17). Ectopic p19 alternative reading frame (ARF) expression tumorigenesis (8). Antiproliferative agents such as endostatin (9) and stabilizes p53 and induces p53-responsive genes, including the pharmaceutical agents with antiproliferative properties including acy- p21Cip1 gene that contributes to cell cycle arrest in fibroblasts (28). Ј clic retinoid, flavoperidol, and phosphatidylinositol 3 -kinase inhibi- The molecular targets of INK4a/ARF in mammary epithelial cells are tors repress cyclin D1 transcription and abundance leading to cell not well understood, although loss of p16INK4a in breast cancer cells contributes to extended growth capacity (29), and reintroduction of Received 8/13/03; revised 3/30/04; accepted 4/29/04. p16INK4a into breast cancer epithelial cells induces a dose-dependent Grant support: This work was supported in part by awards from the Susan Komen Breast Cancer Foundation, Breast Cancer Alliance Inc., Grants R01CA70896, cell cycle arrest (30, 31). Breast cancer cell lines are deleted of R01CA75503, R01CA86072, R01CA86071 (to R. G. Pestell), and R03AG20337 (to C. p16INK4a in 40–60% of cases (32), and DNA methylation, an alter- Albanese). R. G. Pestell was a recipient of the Irma T. Hirschl and Weil Caulier award and nate mechanism of p16INK4a inactivation, has been reported to occur was the Diane Belfer Faculty Scholar in Cancer Research. M. D’Amico was a recipient of the New York State mentored EMPIRE award. Work conducted at the Lombardi Cancer in up to 30% of human breast cancers (33, 34). Reciprocal expression Center was supported by the NIH Cancer Center Core grant. Ink4a/Arf knockout mice of cyclin D1 and p16INK4a has been reported in human breast cancer were a generous gift from Dr. R. DePinho. (34), raising the possibility that p16INK4a may regulate cyclin D1 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with abundance. The current studies investigated the function of Ink4a/Arf 18 U.S.C. Section 1734 solely to indicate this fact. in mammary epithelial cells and the mechanism of cell cycle arrest. Requests for reprints: Department of Oncology, Lombardi Comprehensive Cancer Ϫ/Ϫ Center, Research Building, Room E501, 3970 Reservoir Rd., NW, Box 571468 George- The expression of cyclin D1 is increased in the tissues of Ink4a/Arf town University, Washington, DC 20007. E-mail: [email protected]. mice, and cyclin D1 is a target of transcriptional repression by INK4a and 4122

ARF through distinct DNA sequences in the promoter. As cyclin D1 abundance is rate- limiting in breast epithelial cell proliferation and tumorigenesis, INK4a and ARF repression of cyclin D1 may contribute to INK4a/ARF tumor suppression function.

EXPERIMENTAL PROCEDURES

Plasmids and Reporter Gene Assays. The luciferase reporter gene con- structions for human cyclin D1, cyclin E, cyclin A, c-Fos, c-Jun, JunB, p16INK4a (6, 35), and expression vectors for p16INK4a, pCMVp16INK4a, pCMV- p16INK4aP114L (36) and pcDNA-p19ARF were described previously. The region from Ϫ1156–1117 was deleted in the context of the Ϫ1745 CD1LUC reporter. Cell culture, DNA transfection, and luciferase assays were performed as described previously (35). The MCF7 cells were maintained in MEM with 5% FCS and 1% penicillin/streptomycin. Cells were transfected by calcium phosphate precipitation, the media was changed after 3 h, and luciferase activity determined after a further 24–36 h. Comparison was made between the effect of transfecting active expression vector with the effect of an equal amount of vector cassette. At least three different plasmid preparations of each construct were used. In each experiment, a dose response was determined with 150, 300, or 600 ng of expression vector as indicated in the figure legend (3,4,6,8) and

Fig. 2. p16INK4a and p19ARF decrease S-phase in MCF7 cells. Flow cytometric analysis of propidium iodide-stained MCF7 cells transfected and magnetic activated cell sorted with the expression vectors for p16INK4a, p16INK4aP114L, or p19ARF as indicated. ARF, alternative reading frame; INK, inhibitor of cyclin-dependent kinase.

the cyclin D1 promoter reporter plasmids (4.8 ␮g). Luciferase assays were performed at room temperature using an Autolumat LB 953 (EG&G Berthold), and the initial 30 s of the reaction were used to assess luciferase content with the values expressed in arbitrary light units. Background activity from cell extracts was typically Ͻ100 Absolute Light Units/10 s. Statistical analyses was performed using the Mann-Whitney U test, and significant differences were established as P Ͻ 0.05. Antibodies, Reagents, and Northern and Western Blot Analysis. MCF7 cells were transfected with empty vector or expression plasmids encoding p16INK4a or p19ARF along with pMACS4.1 and sorted for transfection using an autoMACS cell sorter (Miltenyi Biotech, Auburn, CA). Cellular extracts were then processed for either Northern or Western blot analysis as described- deficient previously in this laboratory (11, 35). Tissues isolated from Ink4a/ Arf-deficient mice, and cells were lysed in buffer containing 50 mM HEPES (pH 7.2), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.1% Tween Fig. 1. Increased cellular proliferation and cyclin D1 abundance in Ink4a/Arf-deficient 20, 0.1 mM phenylmethylsulfonyl fluoride, 2.5 mg/ml leupeptin, and 0.1 mM Ϫ Ϫ cells. A and B, mouse embryonic fibroblast cells were isolated from Ink4aArf / or Na3VO4. Total protein was determined using the Bradford method (Bio-Rad, littermate control mice. Cells were plated at 50,000 cells/60-mm dish. Dishes were Hercules, CA). Lysates (100 ␮g) were separated on 12% SDS-PAGE and incubated at 37°C, and cells were counted at 1-day intervals using a hemocytometer. Western blot for p16INK4a is shown. The data are shown as mean Ϯ SE for N ϭ 3 separate blotted onto nitrocellulose. Membranes were blocked with 5% milk in PBS- experiments. INK, inhibitor of cyclin-dependent kinase; KO, knockout; HET, heterozy- 0.2% Tween 20 for1hatroom temperature before exposure of primary ϩ Ϫ gous. B, data are shown at a high resolution for proliferation rates of Ink4a/Arf / mouse antibodies to cyclin D1 (AB-3; NeoMarkers, Fremont, CA), p16INK4a, p19ARF, embryonic fibroblasts. C, increased cyclin D1 abundance in Ink4a/Arf-deficient tissues. ϩ Ϫ Ϫ Ϫ p53, activating transcription factor-2 (ATF-2), cAMP-responsive element- Western blotting of tissue from Ink4a/Arfwt, Ink4a/Arf / ,orInk4a/Arf / for cyclin D1 and the loading control GDI. ARF, alternative reading frame; wt, wild type; GDI, guanine binding protein (CREB; Santa Cruz Biotechnology, Santa Cruz, CA), or dissociation inhibitor. guanine dissociation inhibitor (a gift from Dr. P. Bickel). Membranes were 4123

Fig. 3. The cyclin D1 promoter is repressed by p16INK4a in MCF7 breast epithelial cells. A and B, luciferase activity of the cyclin D1 promoter (Ϫ1745 CD1LUC; 4.8 ␮g) and either viral, immediate early gene, or cell cycle control promoters were determined in MCF7 cells transfected with expression vectors for p16INK4a (control, f; 150, Ⅺ; 300, u; and 600 ng, u). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; INK, inhibitor of cyclin-dependent kinase; LUC, luciferase. Inset, the data are shown as mean Ϯ SE for N Ͼ 8 separate experiments. Fold change is shown, compared with the effect of equal amounts of empty expression vector cassette for the effect of the p16INK4a expression vector. Northern blot analysis of MCF7 cells transfected with p16INK4a and subjected to magnetic activated cell sorting of transfected cells. C, comparison of cyclin D1 promoter repression by either p16INK4a wild type or the cdk-binding-defective p16INK4a mutant. The data are mean Ϯ SEM of N ϭ 8 separate experiments.

washed extensively in PBS-0.2% Tween 20 and incubated with appropriate charcoal stripped dextran serum for 3 days. Upon estradiol (100 nM) stimula- secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Bio- tion for 45 min, the cells were cross-linked by adding 1.1% formaldehyde technology). Membranes were washed extensively with PBS-0.2% Tween 20, buffer containing 100 mM sodium chloride, 1 mM EDTA-Na (pH 8.0), 0.5 mM and proteins were visualized using chemiluminescence. Fold increase in pro- EGTA-Na, and Tris-HCl (pH 8.0) directly to culture medium for 10 min at tein levels were plotted against a standard of 293T cells transfected with the expression vector for the relevant protein. Flow Cytometric Analyses. Selection of transfected cells using the CD4 as a marker was performed as described previously using magnetic activated cell sorting (MACS; Ref. 37). Flow cytometric analyses were carried out in a fluorescence-activated cell sorter (FACStar plus; Becton Dickinson) with a 360–365 nM argon-iron laser (38). Histograms were analyzed for cell cycle compartments using ModFit version 2.0 (Verity Software House, Topsham, ME). To select transfected cells, cotransfection experiments were conducted using magnetic separation of transfected cells using CD4 as the marker and the MACS. A comparison was made between cells transfected with vector encoding p16INK4a, p19ARF expression plasmid, and cells transfected with empty expression vector cassette. Cells were stained with cell-permeable DNA- binding dye Hoechst 33342 (10 mg/ml) for 2 h before harvesting, and all subsequent solutions contained Hoechst 33342. Cells were harvested 48 h after transfection using EDTA (5 mg/ml) in PBS. Cells were pelleted by centrifu- gation and resuspended in PBS containing anti-CD4-coated magnetic beads. Cells were incubated with beads for 20 min, and CD4-positive cells were obtained using a separating column. Cell cycle analysis was performed in a flow cytometer with a 360–365 nM argon-iron laser. Western blotting was performed on the cells as described above (39). Ink4a/Arf؊/؊ Mice. The Ink4a/Arf knockout mice, with portions of exons 2 and 3 replaced by a neo cassette (40), were established in the Friends virus B strain through 11 back-crossings. Mice were screened by PCR for the Ink4a/Arf genomic status. DNA was extracted by standard methods from a small piece of tail tissue cut from each animal at the time of weaning (41). For evaluating the Ink4a/Arf status of offspring, PCR reactions using three primers allowed for simultaneous detection of both the normal and mutant p16INK4a allele in a single reaction. These primers consisted of a common 5Ј sense primer, p16–1F (5ЈTCCCTCTACTTTTTCTTCTGAC-3Ј); two 3Ј antisense primers, p16–1R (5ЈCGGAACGCAAATATCGCAC-3Ј); and p16-Nul (5Ј- CTAGTGAGACGTGCTACTTC-3Ј). Reactions were run for 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Mouse embryo fibroblasts were prepared from Ink4a/ArfϪ/Ϫ or wild-type litter mate controls as described previously (11). Fig. 4. Repression of the cyclin D1 promoter by p16INK4a. Luciferase activity of the Ϫ Chromatin Immunoprecipitation (ChIP) Assay. ChIP analysis was per- cyclin D1 promoter ( 1745 CD1LUC) and the viral or cell cycle control gene promoters (4.8 ␮g) were assessed in 293T cells in the presence or absence of p16INK4a (control, f; formed following a protocol provided by Upstate Biotechnology under mod- 150, Ⅺ; 300, u; and 600 ng, u). INK, inhibitor of cyclin-dependent kinase; CMV, ified conditions. MCF7 cells (1 ϫ 106) were grown in DMEM with 10% cytomegalovirus; LUC, luciferase; HEK, human embryonic kidney. 4124

Fig. 5. The cyclin D1 promoter ATF/CRE site is required for p16INK4a repression in MCF7 cells. A, luciferase activity from either the wild type (wt; Ϫ1745CD1LUC; 4.8 ␮g) or the cyclin D1 promoter luciferase reporters with point mutations of the TCE or CRE site was compared for repression by p16INK4a (300 ng) in either MCF7 or (B) SKBR3 cells. The data are shown as mean Ϯ SEM for N Ͼ 8 separate experiments. INK, inhibitor of cyclin-dependent kinase; LUC, luciferase; TCE,T cell factor. C, relative luciferase activity of the Ϫ1745CD1LUC (4.8 ␮g) was compared in cells cotransfected with either cAMP-responsive element-binding protein or ATF-2 wt or dominant- negative mutant expression vector using either 300 (u) or 600 ng (u). Comparison was made between the effect of the wt or dominant-negative vector and equal amounts of empty expression vector cassette. MCF7 cells transfected and magnetic activated cell sorted with the empty control vector and expression vector for p19ARF. Western blot of transfected cells are shown. Antibodies were used as shown. ATF, activating transcription factor; CRE, cAMP-response element; ARF, alternative reading frame; GDI, guanine dissociation inhibitor; DN, dominant-negative; DBD LZ, DNA-binding domain leucine zipper.

37°C. The medium was aspirated, washed thrice using ice-cold PBS containing CTGGGACAAG-3Ј and 5Ј-GACCCTCTCATGTAACCACG-3; primer pair 10 mM DTT and protease inhibitors, lysed by warm 1% SDS lysis buffer, and for AP-1 site, 5Ј-CTGCCTTCCTACCTTGACCA-3Ј and 5Ј-TGAAGG- incubated for 10 min on ice. The cell lysates were fornicated to shear DNA to GACGTCTACACCCC-3Ј; primer pair for E2F site, 5Ј-CTCCACCTCAC- lengths between 200 and 1000 bp, and thee samples were diluted to 10-fold in CCCCTAAAT-3Ј and 5Ј-GGGGGCGGGCGCAGGGGG-3Ј; primer pair for ChIP dilution buffer. To reduce nonspecific background, cell pellet suspension cAMP-response element (CRE) site, 5Ј-GCCCCCCTCCCGCTCCCATT-3Ј was precleared with 60 ␮l of salmon sperm DNA/protein-A agarose-50% and 5Ј-TGGGGCTCTTCCTGGGCAGC-3Ј. slurry (Upstate Biotechnology) for2hat4°C with agitation. Chromatin MCF7 cells were transiently transfected with either cyclin D1 Ϫ1745 or solutions were precipitated overnight at 4°C using 4 ␮g of anti-p53 (FL-393; cyclin D1 Ϫ1745-ARF-response element deletion mutant reporter followed by Santa Cruz Biotechnology) with rotation. For a negative control, rabbit IgG treatment with estradiol for 45 min. The cells were cross-linked with formal- was immunoprecipitated by incubating the supernatant fraction for1hat4°C dehyde buffer for 10 min at 37°C and followed the procedure as mentioned with rotation. Sixty ␮l of salmon sperm DNA/protein-A agarose slurry was above. added for2hat4°C with rotation to collect the antibody/histone complex and washed extensively following the manufacturer’s protocol. Input and immu- RESULTS noprecipitated chromatin were incubated at 65°C overnight to reverse crosslinks. After proteinase K digestion for 1 h, DNA was extracted using Increased Cyclin D1 Abundance in Ink4a/Arf Deficient Cells. Qiagen spin column kit. Precipitated DNAs were analyzed by PCR. ChIP The p16INK4a tumor suppressor was initially defined through its analysis was conducted of either endogenous gene promoters or in a subset of ability to inhibit Ras-induced transformation (42) and embryonic experiments, MCF-7 cells were transiently transfected with plasmid DNA- Ϫ/Ϫ encoding promoter reporter gene constructs, and ChIP analysis was conducted fibroblasts from Ink4a/Arf mice [mouse embryonic fibroblast using a reduction in the number of cycles from 30 to 25. The following primers (MEFs)] exhibit enhanced cellular proliferation. Conversely, cyclin were used for PCR: for mdm2 promoter, 5Ј-TGGGCAGGTTGACT- D1-deficient mice are resistant to Ras-induced transformation and Ϫ/Ϫ CAGCTTTTCCTC-3Ј and 5Ј-TGGCGTGCGTCCGTGCCCAC-3Ј; for cyclin cyclin D1 MEFs show reduced cellular proliferation (42). The D1 promoter, primer pair for ARF-response element site, 5Ј-TCCCCTCTGC- transcriptional targets of p16INK4a mediating tumor suppressor func- 4125

Fig. 6. A novel p19ARF-response element required for repression of the cyclin D1 promoter by p19ARF in MCF7 cells. A, luciferase activity of the cyclin D1 promoter (Ϫ1745 CD1LUC; 4.8 ␮g) and the viral or cell cycle control gene promoters were assessed in MCF7 cells in the presence or absence of p19ARF (control, f; 150, Ⅺ; 300, u; 600 ng, u). The data are shown as mean Ϯ SE for N Ͼ 8 separate experiments. B, luciferase activity from either the wild type (Ϫ1745CD1LUC) or the cyclin D1 promoter luciferase reporters with point mutations of the CRE, TCF, or AP-1/CRE sites or the (C) mutation of the candidate ARF-response element (ARE) were compared for repression by p19ARF (300 ng) in MCF7 cells. The data are shown as mean Ϯ SE for N Ͼ 8 separate experiments. Comparison was made between the effect of the p19ARF expression vector and equal amounts of empty expression vector cassette. CRE, cAMP-response element; ARF, alternative reading frame; LUC, luciferase; TCF, T cell factor.

tion and inhibition of DNA synthesis remain to be fully defined. p16INK4a repressed the human cyclin D1 promoter, in a dose-depen- Oncogenic Ras was previously shown to induce cyclin D1 in rodent dent manner (Fig. 3A) and reduced cyclin D1 protein levels 40% (data fibroblasts and human trophoblast cells (2). We investigated the not shown). To assess further the mechanism of cyclin D1 promoter possibility that cyclin D1 may function as a Ras-inducible gene that is transcriptional repression by p16INK4a, the promoters of several cell a target of p16INK4a repression. cycle control genes were assessed. p16INK4a did not inhibit the activity Consistent with previous studies, the proliferation rates of Ink4a/ of the human c-fos promoter, consistent with a previous study in ArfϪ/Ϫ MEFs were increased compared with litter mate controls (Fig. which p16INK4a failed to repress the serum-response element of the ϩ/Ϫ 1A) and Ink4a/Arf MEF proliferation rate was greater than Ink4a/ c-fos promoter (42). The pA3LUC vector reporter was unaffected by ArfϪ/Ϫ MEFs (Fig. 1B). To determine whether reduced p16INK4a p16INK4a, indicating that the effect was not mediated by a general levels were associated with increased abundance of cyclin D1 in vivo, effect on luciferase activity (Fig. 3A). The viral cytomegalovirus we examined the tissues of mice genetically deficient for Ink4a/ (CMV) promoter was induced 3-fold by p16INK4a. Arfϩ/Ϫ mice (40). (These mice were generated by deleting exons 2 The cyclin E promoter was repressed by p16INK4a (Fig. 3A); how- and 3 of the Cdkn2a locus and are functional knockouts for both ever, the cyclin A promoter was unaffected, and the p21CIP1/WAF1 p16INK4a and p19ARF genes, which share exons 2 and 3 of the Cdkn2a promoter was induced; thus p16INK4a conveys promoter-specific reg- locus). Compared with the Ink4a/Arf wt mice, the Ink4a/Arf-deficient ulatory effects. The p16INK4a mutant defective in binding cdk4 mice tissues showed 2- to 3-fold increased abundance of cyclin D1 (p16INK4a-P114L; Ref. 36) repressed basal cyclin D1 promoter activ- protein (Fig. 1C). The cytoplasmic protein, guanine dissociation in- ity to a similar degree as p16INK4a wt (Fig. 3B). Thus the mechanism hibitor, used as a loading control, showed similar abundance of by which p16INK4a represses basal activity of the cyclin D1 promoter protein between genotypes. appears to be independent of its cdk4-binding function. Because p16INK4a and p19ARF Inhibits DNA Synthesis and Apoptosis in p16INK4a failed to repress cyclin A, which is induced during S-phase MCF7 Breast Cancer Epithelial Cells. To investigate the mecha- entry, and the cdk4-binding-defective mutant repressed the cyclin D1 nisms by which p16INK4a or p19ARF regulate DNA synthesis, MCF7 promoter, these findings raise the possibility that the effect of cells were transfected with expression vectors for either p16INK4a or p16INK4a on cyclin D1 promoter activity is not mediated indirectly p19ARF and subjected to MACS sorting with subsequent fluorescence- through effects on DNA synthesis. activated cell sorter analysis and Western blotting. p16INK4a reduced Cyclin D1 abundance regulates MCF7 cell cycle progression, mak- S-phase by 40% (Fig. 2), consistent with previous studies (31), and ing a determination of the effect of p16INK4a independent of cell cycle p19ARF reduced the S-phase fraction by 30%, consistent with previous complex. The ability of cyclin D1 to induce cell cycle progression is studies in fibroblasts (43). dependent upon the presence of the pRB protein, which is phospho- Selective Repression of Cyclin D1 Promoter by p16INK4a. rylated and inactivated by cyclin D1 kinase activity (44, 45). pRB is Northern blot analysis of transfected and MACS-sorted MCF7 cells inactivated by E1A and large T antigen (46). We therefore assessed showed that p16INK4a reduced cyclin D1 mRNA (Fig. 3A, right). the effect of p16INK4a on transcriptional regulation of the cyclin D1 4126

reduced ATF-2 and CREB protein 40–50%. Together these studies indicate that CREB/ATF-2 protein expression regulates basal cyclin D1 abundance in MCF7 cells, and p16INK4a repression of cyclin D1 involves the ATF-2/CRE site in the cyclin D1 promoter in breast cancer epithelial cells. Repression of Cyclin D1 by p19ARF Functions through a Novel Distal Response Element. The reciprocal regulation of cyclin D1 and p16INK4a abundance has been observed in human breast cancers (34, 47) and in the Ink4a/ArfϪ/Ϫ mice mammary gland (48, 49). The Ink4a/ArfϪ/Ϫ mice are deficient in both p16INK4a and p19ARF protein. We therefore examined the role of p19ARF in regulating cyclin D1 promoter activity in MCF7 cells. p19ARF repressed cyclin D1, the cyclin A and p21CIP1 promoters in a dose-dependent manner, without affecting the activity of the viral CMV-LUC reporter (Fig. 6A). Mutation of the Tcf site or the AP-1 and CRE site also did not reduce repression but rather enhanced repression by p19ARF. p19ARF transfection of MCF7 cells induced p53 and reduced cyclin D1 protein levels consistent with previous studies that p19ARF functions in a genetic pathway that involves p53 (Refs. 49, 50; data not shown). Mutation of the ATF/CRE site that had abolished repression by p16INK4a did not affect repression by p19ARF (Fig. 6B). p53 is capable of regulating gene expression either directly through consensus p53- response elements or through atypical response elements upon physical interaction with other proteins including WT1-Egr and nuclear receptors (51–53). We scrutinized the sequences of the cyclin D1 promoter for DNA sequences that could potentially respond to p53 either directly or indirectly. In the absence of a consensus p53- response element, we mutated elements resembling an atypical p53- response element in the cyclin D1 promoter at Ϫ1137 (54). p19ARF- mediated repression was abrogated by deletion of the sequences, strongly suggesting a key role for this element in p19ARF repression of cyclin D1 transcription (Fig. 6C). To further examine the role of p53 in the repression of the cyclin Fig. 7. ChIP assays reveal p53 is bound to the cyclin D1 promoter. A, MCF7 cells were ARF subjected to chromatin immuunoprecipitation assays. PCR using oligonucleotides flank- D1 promoter by p19 , we performed ChIP assays in MCF7 cells. ing the cyclin D1 ARF-response element (ARE), and E2F site (E2F) revealed a band in Oligonucleotides were generated for PCR amplification of the p53- samples immunoprecipitated with antibodies to p53 corresponding to the endogenous cyclin D1 promoter. CRE, cAMP-response element; LUC, luciferase. B, MCF7 cells response element of the mdm2 promoter as a form of positive control transfected with a cyclin D1 promoter construct also indicated that p53 was bound to the for the ChIP assays and to the cyclin D1 promoter CRE site, E2F site, ARF-response element (ARE) and E2F sites. C, p53 was not bound in MCF7 cells and the ARF-response element. p53 bound the mdm2 promoter p53- transfected with a mutant cyclin D1 promoter construct. response element in ChIP assays but was not identified at the cyclin D1 promoter CRE site. In contrast, p53 was contained within ChIP promoter in E1A and large T antigen transformed 293T cells. Cyclin assays of the cyclin D1 promoter ARF-response element and E2F site D1 promoter activity was repressed by p16INK4a (Fig. 4A), while (Fig. 7A). Transfected cyclin D1 promoter recruited p53 to the ARF- inducing the activity of the c-fos,c-jun, cdc25a, and p21Cip1/WAF1 response element but did not bind p53 at the CRE site. Mutation of the promoters. In contrast the CMV promoter was unaffected, and the cyclin D1 promoter ARF-response element abrogated p53-binding to cyclin E promoter was repressed (Fig. 4, A and B). the promoter in ChIP assays, strongly suggesting this site is required p16INK4a Repression of the Cyclin D1 Promoter Requires the for p53 binding to the cyclin D1 promoter (Fig. 7). CRE/ATF-2-Binding Site in MCF7 Breast Epithelial Cells. Ex- To determine whether p19ARF repression of cyclin D1 transcription pression of cyclin D1 is induced by diverse signaling pathways in was unique to MCF7 cells, we assessed transcriptional regulation in MCF7 cells including Ras, Wnt, and ErbB2 through distinct DNA the embryonal kidney cell line, 293T. Cyclin D1 and cyclin A pro- sequences including a T cell factor (Tcf), E2F, and ATF/CRE site. moter activity was repressed in a dose-dependent manner by p19ARF, Point mutation of the ATF/CRE site, but not the Tcf site, abrogated without affecting CMV promoter activity (Fig. 8A). There was a repression and resulted in a promoter fragment that was activated by modest induction of the c-Jun promoter (Fig. 8B). p19ARF repressed p16INK4a (Fig. 5A). Mutation of the ATF/CRE site also abolished the p16INK4a promoter (Fig. 8C) consistent with previous findings that repression by p16INK4a in the human SKBR3 breast cancer cell line expression of p16INK4a is high in ARF-null keratinocytes and bone (Fig. 5B). As the CRE site of the cyclin D1 promoter binds CREB and marrow macrophages (55, 56). 293T cells express E1A and large T, ATF-2 protein in epithelial cells (35, 47), we assessed the role of these functionally inactivating the pRb and p53-response element. Consist- proteins in regulating cyclin D1 promoter activity in MCF7 cells. ent with other studies of ARF target genes, these findings suggest ATF-2 inhibited cyclin D1 promoter activity 35–40%. In contrast, a ARF repression of the cyclin D1 promoter may involve both p53- DNA-binding-defective mutant of ATF-2 that dimerizes with AP-1 dependent and p53-independent signaling mechanisms. Together proteins and functions as a dominant-negative mutant of ATF-2 these studies demonstrate that the cyclin D1 gene is repressed by both function, induced cyclin D1 (Fig. 5C). CREB induced and a domi- reading frames transcribed from the INK4/ARF locus, and transcrip- nant-negative CREB mutant inhibited cyclin D1 promoter activity tional repression is mediated through two distinct DNA sequences (Fig. 5C). MCF7 cells transfected with p16INK4a and MACS sorted (Fig. 8D). 4127

Fig. 8. Repression of the cyclin D1 promoter by p19ARF. A-C, luciferase activity of the cyclin D1 promoter (Ϫ1745 CD1LUC) and the viral or cell cycle control gene promoters (4.8 ␮g) was assessed in HEK293T cells in the presence or absence of p19ARF (control, f; 150, Ⅺ; 300, u; 600 ng, u). The data are shown as mean Ϯ SE for N Ͼ 8 separate experiments. LUC, luciferase; HEK, human embryonic kidney. D, schematic model by which the Ink4a/Arf locus regulates cyclin D1 expression and function. The alternate reading frames encoding p16INK4a and p19ARF repress cyclin D1 promoter activity through distinct DNA sequences. The ARF-response element (ARE) of the cyclin D1 promoter is shown. ARF, alternative reading frame; INK, inhibitor of cyclin-dependent kinase.

DISCUSSION cycle arrest that correlates with a reduction in pRb phosphorylation corresponds to a repression of E2F-regulated genes (36, 63, 64). In the The current studies provide evidence that the cyclin D1 gene is current studies, the E2F-responsive elements of the cyclin D1 pro- under transcriptional control by both components of the INK4a/ARF moter were not involved in the transcriptional repression by p16INK4a locus. Increased cyclin D1 protein abundance was observed in several and p19ARF. Together these findings suggest the mechanism by which Ink4a/Arf-deficient tissues, and both p16INK4a and p19ARF inhibited p16INK4a represses cyclin D1 transcription described herein is distinct cyclin D1 abundance in MCF7 cells. The increased abundance of from previous studies in which p16INK4a regulated expression of cell cyclin D1 in Ink4a/Arf-deficient tissues is consistent with recent cycle control genes indirectly through binding cdk4 and inhibiting findings of increased cyclin D1 levels in mammary tumors of trans- cyclin D1 kinase activity. genic mice induced by ErbB2 that were Ink4a/Arf-deficient (49). The p16INK4a and p19ARF inhibited cyclin D1 promoter activity through Ink4a/Arf deletions (23, 57, 58), like cyclin D1 overexpression, cor- distinct DNA sequences. The CRE/ATF-2 site of the cyclin D1 relate with poor outcome from tumors in patients and in mice. As the promoter was required for repression by p16INK4a in MCF7 and abundance of cyclin D1 is rate limiting in G1 phase progression in mammary epithelial cells (7), regulation of cyclin D1 abundance by SKBR3 cells. In our previous studies, electrophoretic mobility shift p16INK4a and p19ARF may contribute to the cell cycle control or tumor assays identified CREB and ATF-2 as the dominant proteins binding suppressor functions of these proteins. the cyclin D1 CRE site in MCF7 cells (65). ATF-2, which binds the Although p16INK4a was originally identified as a protein binding to CRE site with CREB in MCF7 cells (65), repressed cyclin D1, raising cdk4 that acts upstream of Rb to cause G arrest (59), cdk4-binding the possibility that ATF-2 may play an intermediary role in repression 1 INK4a independent functions of p16INK4a contribute to cell cycle arrest (60, of cyclin D1 by p16 . Herein, transfection of wild-type CREB 61). Several lines of evidence suggest the mechanism of transcrip- enhanced cyclin D1 promoter activity, and a dominant-negative mu- tional repression of the cyclin D1 promoter by p16INK4a is specific and tant CREB repressed promoter activity 15–20%. A dominant-negative v-src not an indirect effect of the expressed proteins on cell cycle control. CREB was shown previously to inhibit pp60 -induced cyclin D1 First, p16INK4a displayed promoter-selective function and was capable expression in MCF7 cells and point mutation of Ser-133 in CREB was v-src of either repressing (cyclin D1, Jun B) or activating (c-jun, cyclin A) shown to abolish induction by pp60 . The CREB coactivator, CBP, transcription of specific target genes. Second, repression by p16INK4a is tethered to the basal transcription apparatus and RNA polymerase II displayed sequence-selective function. Third, a cdk4-binding-defec- by RNA helicase A (66). The RNA polymerase II carboxyl-terminal tive mutant of p16INK4a repressed the cyclin D1 promoter. Fourth, domain is activated and phosphorylated by TFIIH, which contains p16INK4a repressed cyclin D1 in E1A and large T overexpressing cdk7. Furthermore, p16INK4a inhibits carboxyl-terminal kinase activ- 293T cells. As pRb is inactivated by E1A and large T antigen (46), an ity (61). Together these studies raise the possibility that p16INK4a has indirect effect of p16INK4a on cyclin D1 through the pRB protein (44, the capacity to repress CRE activity of the cyclin D1 promoter 45) seems less likely. Fifth, the cyclin D1 gene is regulated by pRb through interactions with components of the basal transcription appa- and E2F/DP proteins through an E2F binding site (6, 62). Studies with ratus. Several oncogenic signaling pathways activate cyclin D1 ex- dominant-negative E2F mutants demonstrated that the cyclin D1 E2F pression through the CRE site, including SV40 small t antigen (67) sites are functional in MCF7 cells (6). The p16INK4a-induced cell and activating mutants of pp60v-src (65). Together, these observations 4128

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. The Inhibitor of Cyclin-Dependent Kinase 4a/Alternative Reading Frame ( INK4a/ARF) Locus Encoded Proteins p16 INK4a and p19ARF Repress Cyclin D1 Transcription through Distinct cis Elements

Mark D'Amico, Kongming Wu, Maofu Fu, et al.

Cancer Res 2004;64:4122-4130.

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