[CANCER RESEARCH 63, 958 –964, March 1, 2003] Peroxisome Proliferator-activated Receptor Agonists Induce Proteasome-dependent Degradation of Cyclin D1 and Estrogen Receptor in MCF-7 Breast Cancer Cells 1 Chunhua Qin, Robert Burghardt, Roger Smith, Mark Wormke, Jessica Stewart, and Stephen Safe 2 Departments of Veterinary Physiology and Pharmacology [C. Q., M. W., J. S., S. S.], Veterinary Anatomy and Public Health [R. B.], and Veterinary Pathobiology [R. S.], Texas A&M University, College Station, Texas, and Institute of Biosciences and Technology, The Texas A&M University System Health Science Center, Houston, Texas 77843-4466 [S. S.] ABSTRACT Treatment of MCF-7 cells with the peroxisome proliferator-activated receptor (PPAR) agonists ciglitazone or 15-deoxy-12,14-prostaglandin J2 resulted in a concentration- and time-dependent decrease of cyclin D1 and estrogen receptor (ER) proteins, and this was accompanied by decreased cell proliferation and G 1 -G 0 3S-phase progression. Down- regulation of cyclin D1 and ERby PPARagonists was inhibited in cells cotreated with the proteasome inhibitors MG132 and PSII, but not in cells cotreated with the protease inhibitors calpain II and calpeptin. Moreover, after treatment of MCF-7 cells with 15-deoxy-12,14-prostaglandin J2 and immunoprecipitation with cyclin D1 or ERantibodies, there was enhanced formation of ubiquitinated cyclin D1 and ERbands. Thus, PPAR-induced inhibition of breast cancer cell growth is due, in part, to proteasome-dependent degradation of cyclin D1 (and ER), and this pathway may be important for other cancer cell lines. INTRODUCTION Peroxisome proliferators were initially characterized from a large group of synthetic industrial and pharmaceutical chemicals that in- duced hepatic hypertrophy and hyperplasia in rodents (1–3). The effects of these compounds were accompanied by increased numbers and size of liver peroxisomes and induction of enzymes required for oxidative metabolism of fatty acids and members of the cytochrome P4504A (CYP4A) family. The intracellular receptor for peroxisome proliferator-induced hepatic responses was first reported in 1990 as PPAR 3 (4), and subsequent studies in several laboratories have also characterized PPAR(or PPAR), PPAR, and several isoforms that arise from alternative splicing and promoter use (5–9). PPARs are dif- ferentially expressed in various tissues and tumors and play a critical role in fatty acid metabolism and energy homeostasis (reviewed in Refs. 1–3). PPARs are ligand-activated transcription factors and members of the nuclear receptor superfamily (10, 11). Activation of PPARs is a multistep process that involves ligand binding and hetero- dimerization with the retinoic X receptor, interaction with sequence- specific gene promoter elements, and recruitment of coactivators and other nuclear coregulatory proteins. PGJ2 is the most potent eico- sanoid activator of PPAR(12, 13); thiazolidinediones such as cigli- tazone are synthetic PPARagonists used extensively for their an- tidiabetic properties and treatment of insulin-resistant type II diabetes (14 –17). PPARis widely expressed in multiple tumors and cell lines, and this receptor has also become a target for developing new anticancer drugs that will take advantage of the antiproliferative effects mediated through PPAR. For example, a recent study investigated PPAR expression in 339 clinical tumor samples from colon, breast, lung, prostate, osteosarcomas, glioblastomas, acute myelogenous leukemia, adult T-cell leukemia, B-cell acute lymphoblastic leukemia, B-cell non-Hodgkin’s lymphoma, and myelodisplastic syndrome (18). Wild- type PPARmRNA was expressed in all tumor specimens, and receptor mutants were not detected in any of these samples. The growth-inhibitory effects of endogenous and synthetic PPARago- nists have been investigated in several tumors and cancer cell lines (19 – 42), and a number of these studies show that ligands for this receptor induce apoptosis and/or decrease G 0 -G 1 3 S-phase cell cycle progression, which is accompanied by a decrease in cyclin D1 or modulation of cdk inhibitors and other factors involved in cell growth. Studies in breast cancer cells show that PPARagonists inhibit growth of ER-positive and -negative cell lines. Treatment of ER- positive MCF-7 cells with PPARagonists inhibits activation of epidermal growth factor receptors through inhibition of tyrosine phos- phorylation (42) and up-regulates PTEN expression in MCF-7 and other cancer cell lines (41). PGJ2 also repressed cyclin D1 mRNA and protein in MCF-7 cells, and inhibition of transactivation was associ- ated with enhanced recruitment of limiting cellular levels of p300 to PPAR(39). This study further investigates the mechanism of PPAR-induced inhibition of cancer cell growth using MCF-7 human breast cancer cells as a model. The results show that both PGJ2 and ciglitazone (a thiazolidinedione) induce proteasome-dependent degra- dation of cyclin D1 and ER, and this represents a novel pathway for PPAR-mediated growth arrest in breast cancer cells and is consistent with their inhibition of G 0 -G 1 3 S-phase progression. MATERIALS AND METHODS Cells, Chemicals, Biochemicals, and Other Materials. MCF-7 cells were obtained from American Type Culture Collection (Manassas, VA) and main- tained in MEM with phenol red and supplemented with 0.22% sodium bicar- bonate, 10% FBS, 0.011% sodium pyruvate, 0.1% glucose, 0.24% HEPES, 10 -6 % insulin, and 10 ml/liter antibiotic solution. Cells were grown in 150- cm 2 culture plates in an air:carbon dioxide (95:5) atmosphere at 37°C and passaged every 6 days. Cells were seeded in DMEM:Ham’s F-12 with 5% FBS, and cell proliferation studies were determined using different concentra- tions of PGJ2 or ciglitazone; cell numbers were determined using a Coulter Z1 cell counter. DMSO, PBS, and 100antibiotic solution were purchased from Sigma Chemical Co. (St. Louis, MO). PGJ2 (PG-050) and ciglitazone were purchased from Biomol Research Laboratories Inc. (Plymouth Meeting, PA). MG132, PSII, calpeptin, and CII were purchased from CalBiochem-Novabio- chem Co. (San Diego, CA). FBS was obtained from Intergen (Purchase, NY). Horseradish peroxidase substrate for Western blot analysis was purchased from New England Nuclear Life Science Products (Boston, MA). Antibodies for cyclin D1 (sc-718 and sc-246), ER(sc-544 and sc-8005 for Western blot and immunoprecipitation, respectively), PPAR(sc-7196), Sp1 (sc-59 and sc-420), ubiquitin (sc-8017), cdk4 (sc-260), and preimmune IgG were pur- chased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoprecipita- Received 5/14/02; accepted 12/27/02. 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 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grant ES09106 and the Texas Agricultural Experiment Station. 2 To whom requests for reprints should be addressed, at Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 Texas A&M University, Veterinary Research Building 409, College Station, Texas 77843-4466. Phone: (979) 845- 5988; Fax: (979) 862-4929; E-mail: ssafe@cvm.tamu.edu. 3 The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PGJ2, 15-deoxy-12,14-prostaglandin J2; ER, estrogen receptor; cdk, cyclin-dependent kinase; FBS, fetal bovine serum; -Gal, -galactosidase; TBS, Tris-buffered saline; RIPA, radioimmunoprecipitation assay; WCL, whole cell lysate; PI, propidium iodide; AhR, aryl hydrocarbon receptor; CII, calpain II. 958 Research. on April 9, 2017. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from