Histone Deacetylase Inhibitor Trichostatin A Represses Estrogen Receptor -Dependent Transcription and Promotes Proteasomal Degradation of Cyclin D1 in Human Breast Carcinoma Cell Lines John Patrick Alao, 1 Eric W-F. Lam, 1 Simak Ali, 1 Laki Buluwela, 1 Walter Bordogna, 2 Peter Lockey, 2 Rana Varshochi, 1 Alexandra V. Stavropoulou, 1 R. Charles Coombes, 1 and David M. Vigushin 1 1 Department of Cancer Medicine, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom; and 2 Argenta Discovery Ltd., Harlow, United Kingdom ABSTRACT Purpose: Estrogen receptor  (ER)-positive breast cancer cell lines are up to 10 times more sensitive than ER-negative cell lines to the antiproliferative activity of the histone deacetylase inhibitor trichostatin A (TSA). The pur- pose of the study was to investigate the mechanisms under- lying this differential response. Experimental Design and Results: In the ER-positive MCF-7 cell line, TSA repressed ER and cyclin D1 tran- scription and induced ubiquitin dependent proteasomal deg- radation of cyclin D1, leading primarily to G 1 -S-phase cell cycle arrest. By contrast, cyclin D1 degradation was en- hanced but its transcription unaffected by TSA in the ER- negative MDA-MB-231 cell line, which arrested in G 2 -M phase. Cyclin D1 degradation involved Skp2/p45, a regula- tory component of the Skp1/Cullin/F-box complex; silencing SKP2 gene expression by RNA interference stabilized cyclin D1 and abrogated the cyclin D1 down-regulation response to TSA. Conclusions: Tamoxifen has been shown to inhibit ER-mediated cyclin D1 transcription, and acquired resist- ance to tamoxifen is associated with a shift to ER-inde- pendent cyclin D1 up-regulation. Taken together, our data show that TSA effectively induces cyclin D1 down-regula- tion through both ER-dependent and ER-independent mechanisms, providing an important new strategy for com- bating resistance to antiestrogens. INTRODUCTION Histone deacetylase (HDAC) inhibitors such as the natural antifungal antibiotic trichostatin A (TSA; refs. 1, 2) inhibit the proliferation of tumor cells in culture and in vivo by inducing cell cycle arrest, differentiation and/or apoptosis (3); reviewed in (4). In response to HDAC inhibition, accumulation of hyper- acetylated core histones in chromatin leads to transcriptional activation of certain genes such as the CDKN1A gene, which encodes the p21 WAF1/CIP1 cyclin-dependent kinase inhibitor (5), but leads to transcriptional repression of other genes including the CCND1 gene encoding cyclin D1 (6). D-type cyclins collectively control progression through the cell cycle by activating their cyclin-dependent kinase partners CDK4 and CDK6, which leads to phosphorylation of the reti- noblastoma protein and release of the E2F family of transcrip- tion factors, which, in turn, leads to advancing through G 1 into S phase of the cell cycle (7). Cyclin D1 is strongly implicated in mammary oncogenesis. Cyclin D1 accumulation is normally tightly regulated, but overexpression of the cyclin occurs in some 50% of human breast cancers (8, 9). Cyclin D1 overex- pression is seen in all histologic types of breast cancer and at all stages from carcinoma in situ through metastatic disease but not in premalignant lesions (10, 11). Transgenic mice that overex- press cyclin D1 in mammary tissue develop breast cancers (12) and cyclin D1-deficient mice are resistant to breast cancers induced by neu and ras oncogenes (13). Cyclin D1 may be overexpressed as a result of CCND1 gene amplification or chromosomal translocation (14 –16), sta- bilization of cyclin D1 mRNA (17), or defective degradation of cyclin D1 protein (18). D-type cyclins as well as cyclin E, p21, p27, and E2F-1 are ubiquitinated and targeted for degradation by the 26S proteasome. Phosphorylation of cyclin D1 on thre- onine 286 by glycogen synthase kinase 3 (GSK-3) targets cyclin D1 for ubiquitination (19). Skp2, a regulatory component of the Skp1/Cullin/F-box complex, is implicated in the ubiquiti- nation of cyclin D1; cyclin D1 levels are modestly elevated in Skp2-/- mouse embryo fibroblasts (20), expression of Skp2 antisense induces accumulation of cyclin D1 (20), and defective cyclin D1 degradation in the SK-UT-1B uterine tumor cell line can be rescued by stable transfection of Skp2 but not by a splice variant of Skp2 that does not bind Skp1 and remains in the cytoplasm (21). However, a direct association between Skp2 and cyclin D1 has yet to be confirmed. Estrogen receptor  (ER) is a member of the nuclear receptor superfamily of transcription factors that have highly Received 5/25/04; revised 8/31/04; accepted 9/9/04. Grant support: Supported by Argenta Discovery Ltd. 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. Note: Presented at the 95th Annual Meeting of the American Associa- tion for Cancer Research in Orlando, Florida, March 27–31, 2004. Requests for reprints: David Vigushin, Department of Cancer Medi- cine, 6th Floor MRC Cyclotron Building, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom. Phone: 44-20-8383-8370; Fax: 44-20-8383-5830; E- mail: d.vigushin@imperial.ac.uk. ©2004 American Association for Cancer Research. 8094 Vol. 10, 8094 – 8104, December 1, 2004 Clinical Cancer Research Cancer Research. on December 3, 2021. © 2004 American Association for clincancerres.aacrjournals.org Downloaded from