Effects of oestrogen on gene expression in the epithelium and stroma of the normal human breast

Background

Oestrogen (E2) is central to the development of breast cancer, and anti-oestrogens have been shown to reduce the risk of the disease. However, little is known about the effect of E2 on the normal human breast, particularly when the epithelium and stroma are intact. Previous expression profiles of the response to E2 were performed on tumour cell lines, in the absence of stroma [1–3].

Methods

We investigated gene expression in normal human breast tissue (removed from benign lesions in premenopausal women) transplanted into 9-week-old to 10-week-old athymic nude (Balb/c/nu/nu) mice.

Transplantation was performed in order to obviate the potential effects of the phase of the menstrual cycle. After 2 weeks, when proliferation and progesterone receptor (PR) expression are minimal, the mice were treated with E2 using a 2 mg slow-release pellet for 1 week, which we have previously shown to be optimal for inducing proliferation and PR [4]. At completion of treatment, three tissue samples were generated from each of the six original normal breast tissue samples – two of which were untreated and one of which had been treated with oestrogen. RNA was isolated from each of these samples, then labelled and hybridised to Affymetrix HG-U133A (human) chips on which 22,283 genes are represented. RMA and MAS5 normalisation methods were utilised with bioconductor analysis software.

Results

Oestrogen treatment was found to be the major source of variation in gene expression. Our study shows that known E2-responsive genes such as trefoil factor 1 (pS2) and amphiregulin are also differentially expressed due to oestrogen treatment of normal breast tissue. In addition, many of the genes that showed the greatest responses to E2 have previously been suggested as independent breast cancer prognostic or diagnostic markers (including mammaglobin, prolactin-inducing peptide and keratin 19).

Conclusion

We report the first global gene expression study to look at the effects of oestrogen on the epithelium and stroma of normal human breast tissue, which may give clues to the paracrine action of oestrogen in proliferation. These data form the basis for efforts towards the detection of early gene expression changes leading to breast cancer development.