Methylation of Estrogen and Progesterone Receptor Gene 5’ CpG Islands Correlates with Lack of Estrogen and Progesterone Receptor Gene Expression in Breast Tumors1

Hormonal factors have a profound influence on the development, treatment, and outcome of breast cancer. The absence of steroid hormone receptors is highly correlated with resistance to antihormonal treatments. Work in cultured human breast cancer cell lines has shown that the absence of estrogen receptor (ER) gene expression in ERcells is associated with extensive methylation of the ER gene 5’ CpG island, and treatment with agents that demethylate the ER gene CpG island results in the production of functional ER protein. The current study shows that CpG islands in the 5’ region of the ER and progesterone receptor (PR) genes are methylated in a significant fraction of primary human breast cancer tissues. The ER CpG island is methylated at the methylation-sensitive NotI restriction site in 9 of 39 (25%) of primary ERbreast cancers but remains unmethylated in 53 ER+ breast cancers and 9 normal breast specimens. Three methylation-sensitive restriction sites in the PR gene CpG island are not methylated in normal breast specimens and PR+ human breast cancers but are hypermethylated in 40% of PRhuman breast tumors. These data demonstrate that methylation of the ER and PR gene CpG islands is associated with the lack of ER and PR gene expression in a significant fraction of human breast cancers. Introduction The treatment and outcome of breast cancer are significantly affected by hormonal factors. One-third of breast cancers Received 11/1/95; revised 1/17/96; accepted 1/31/96. 1 This work was supported by American Cancer Society Grants PF4231 (to A. I. F.) and BE237 (to N. E. D.) and the Susan 0. Komen Foundation (to R. G. L.). 2 To whom requests for reprints should be addressed, at The Johns Hopkins Oncology Center, 422 North Bond Street, Baltimore, MD 21231. Fax: (410) 955-0840. lack ER3 and PR, whereas another one-third are ER+ but PR(1). Since the expression of PR requires ER as a transcriptional activator (2), the presence of PR in ER+ tumors is a better predictor for antiestrogen sensitivity than ER alone (1). Because antiestrogens are a first line approach in the treatment of hormone-responsive breast cancer and antiprogestins are currently under investigation, mechanisms of ER and PR gene regulation are critical areas of study. ERtumors lack ER gene expression, but this is not due to mutations within the ER gene (3, 4). PR tumors lack PR transcription, but no mutations within the DNA-binding domain of the ER gene or major polymorphisms or deletions within the PR gene have been identified (5, 6). Acquired loss of transcription of these hormone receptor genes is a potential mechanism of resistance to hormonal therapies. One mechanism by which gene expression can be silenced is aberrant methylation of cytosine-guanine-rich areas termed CpG islands in the 5’ regulatory region and first exon of genes (7). CpG islands are unmethylated in normal adult tissue with the exception of Iranscriptionally silent genes on the inactive X chromosome and some imprinted genes (8, 9). Methylation of these islands has been shown to inhibit transcription directly or stabilize chromatin in a conformation that prevents transcription (10). Work in cultured human breast cancer cells has shown that the absence of ER gene expression in ERcells is associated with increased capacity to methylate DNA and extensive methylation of the CpG island of the ER gene (1 1). Treatment of two human ERbreast cancer cell lines with the demethylating agent, 5-aza-2’-deoxycytidine, results in the demethylation of the ER CpG island and the production of functional ER protein (12, 13). In this study, we determine whether ER CpG island methylation is also observed in primary breast cancers and whether a CpG island identified in the 5’ region of the PR gene could also be altered by DNA methylation. Materials and Methods Cell Lines and Tissue Samples. DNAs from ER+IPR+, ER-/PR, and ER+/PRbreast cancer specimens were obtamed from the Department of Pathology at Vanderbilt University Hospital. Hormone receptor status was analyzed by both biochemical hormone-binding assay and ER immunohistochemical assay as described previously (4, 14). Additional primary breast cancers and reduction mammoplasty specimens were obtained from the Geraldine Brush Cancer Center and the Dcpartment of Medicine at Northwestern University Medical 3 The abbreviations used are: ER, estrogen receptor; ER+ and ER, ER positive and negative, respectively; PR, progesterone receptor; PR+ and PR-, PR positive and negative, respectively. Research. on April 13, 2017. © 1996 American Association for Cancer clincancerres.aacrjournals.org Downloaded from