Endocrine-Related Cancer (2003) 10 193–202 International Congress on Hormonal Steroids and Hormones and Cancer The expression and function of estrogen receptor ␣ and ␤ in human breast cancer and its clinical application S-I Hayashi, H Eguchi, K Tanimoto, T Yoshida, Y Omoto, A Inoue, N Yosida and Y Yamaguchi Division of Endocrinology, Saitama Cancer Center Research Institute, 818 Komuro, Ina-machi, Saitama 362-0806, Japan (Requests for offprints should be addressed to S-IHayashi; Email: [email protected]) Abstract The overexpression of estrogen receptor α (ERα) is frequently observed in the early stage of breast cancer. We previously reported that the specific promoter of the ERα gene is responsible for this enhanced transcription of the gene, and identified the cis-acting elements which play an important role in its transcription. Furthermore, methylation of the ERα gene promoters also contribute to the regulation of gene transcription. Elucidation of these mechanisms of ERα gene expression may provide useful information for the early detection and chemoprevention of breast cancer. On the other hand, the expression of ERβ has been reported in breast cancer. We have also assessed the significance and function of ERβ and its variant types in breast cancer, and suggest that ERβ and ERβcx specifically suppress the function of ERα through different mechanisms. ERβ isoforms may be important functional modulators of the estrogen-signaling pathway in breast cancer cells, and might affect the clinical outcome of patients. Moreover, to address the role of these ERs on the estrogen-dependent growth of breast cancer cells and to develop a diagnostic tool, we have analyzed the gene expression profiles of estrogen-responsive genes using cDNA microarray. Based on these results, the expression of several candidate genes in breast cancer tissues were analyzed by real-time RT-PCR and by immunohistochemical techniques, in order to discover new predictive factors for the endocrine therapy of patients with breast cancer. These studies could provide new clues for the elucidation of the estrogen-dependent mechanisms of cancer and the clinical benefits for patients. Endocrine-Related Cancer (2003) 10 193–202 Introduction in terms of the cancer-specific modulation of ER expression and function, for clinical application. As mentioned above, Estrogen and its receptor (ER) play important roles in the the regulation of ERα gene expression is an important issue α genesis and malignant progression of breast cancer. ER reg- in breast cancer, and the overexpression of ERα is an initial ulates the transcription of various genes as a transcription significant event in its genesis. We have previously reported factor, which binds to estrogen response elements (ERE) that the distal promoter (promoter B) of the ERα gene is α upstream of the target genes. The expression of ER is responsible for this enhanced transcription of the gene closely associated with breast cancer biology, especially the (Hayashi et al. 1997a), and identified an important cis-acting development of tumors; for example, breast carcinomas element, which is located downstream of the transcription which lackER α expression often reveal more aggressive start site and plays an important role in its transcription phenotypes. Furthermore, ERα expression in tumor tissues (Tanimoto et al. 1999). Furthermore, methylation of the ERα is a favorable predictor of prognosis in endocrine treatment. gene promoters also contributes to the regulation of gene Studying the mechanisms that regulate the transcription of transcription (Yoshida et al. 2000). Elucidation of these the ERα gene may therefore provide a new insight into the mechanisms of ERα gene expression is important to develop understanding of breast carcinogenesis. a new tool for the early detection of breast cancer along with We have been investigating the molecular mechanisms chemoprevention targeting breast cancer-specific promoters of carcinogenesis and development of human breast cancer, of the ERα gene (Fig. 1). Endocrine-Related Cancer (2003) 10 193–202 Online version via http://www.endocrinology.org 1351-0088/03/010–193 2003 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/26/2021 04:32:51AM via free access Hayashi et al.: ERα and β in breast cancer estrogen synthesis (aromatase) ? Growth factors ERα gene ? E2 overexpression estrogen ablation LH-RH agonist coactivators aromatase inhibitor phosphorylation ERβ P53 signal AIB1 MDM2 α overexpression ER anti-hormones TAM ROI ERE target genes ICI raloxifene hypoxia redox anti-cancer drugs inflammatory cytokines Figure 1 ERs in the microenvironment of breast cancer. Breast cancer-specific phenomena are illustrated; for example, overexpression of ERα and its coactivators, alteration of intracellular signaling pathways triggered by various stimuli from outside the cancer cells, and the modulation of ERα function by ERβ or cancer-related genes. ROI, reactive oxygen intermediate; TAM, tamofixen; ICI, ICI182780. Modulation of the ER function might be a promising tool There are many reports concerning the target genes tran- with which to control breast cancer. In fact, anti-estrogens scriptionally activated by ERα, but the entire mechanism of are widely used for its therapy. From this aspect, we have the pathway from ERα leading to the proliferation and pro- so far examined the cancer-specific modulation of ERα gression of mammary tumors is far from being completely function, such as redox regulation (Hayashi et al. 1997b) clarified. In order to elucidate the scheme of estrogen sig- and interaction with cancer-related genes (Saji et al. 2001), naling and improvement of clinical decisions, expression pro- while clinically available tools have not been established filing analysis using cDNA microarray technology should be hitherto. one of the most effective procedures. Several laboratories On the other hand, another ER, ERβ, was recently ident- have performed cDNA microarray analysis of breast cancer ified (Kuiper et al. 1996). Subsequently, numerous studies from patients, and novel genes whose expression status was have reported on the expression of ERβ in various cancers, highly correlated with the prognosis of the patients have been including our observations in breast (Omoto et al. 2002), lung identified (Finlin et al. 2001, Sørlie et al. 2001, Veer et al. (Omoto et al. 2001), and stomach (Matsuyama et al. 2002). 2002). There has also been a report concerning gene expres- Immunohistochemical studies suggest that ERβ tends to be sion profiling in human ZR-75-1 breast cancer cells in the expressed in ERα-positive breast cancers, and that there are presence of estrogen or estrogen antagonists using oligo- ERα and ERβ co-expressing cells in human breast cancer. Fur- nucleotide microarray and several novel estrogen-responsive thermore, the existence of various variant forms of ERβ has genes have been identified (Soulez & Parker 2001). Nonethe- been reported in breast cancer cells (Leygue et al. 1999). We less, there is little information on how many markers are suf- then assessed the significance and function of ERβ and its vari- ficient and which markers are suitable for accurate prognosis ant types in breast cancer. Various observations obtained from and diagnosis of breast tumors, especially regarding sensi- experiments using ERβ-expressing stable transformant breast tivity to anti-hormone therapy. cancer cell lines suggested that ERβ and ERβcx truncated at We first analyzed the estrogen-responsive gene expres- the C-terminal region but has extra 26 amino acids (Ogawa et sion profile in ER-positive breast cancer cells using large- al. 1998) specifically suppress the function of ERα through scale cDNA microarray (Inoue et al. 2002). Based on the different mechanisms. These ERβ isoforms could be important results, the custom-made cDNA microarray, on which only functional modulators of estrogen-signaling pathways in estrogen-responsive genes were loaded, was produced. Using breast cancer cells, and might affect the clinical outcome of this microarray consisting of the narrowed gene subset, we patients with primary breast cancer. analyzed the estrogen responsiveness of various cell lines and 194 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/26/2021 04:32:51AM via free access Endocrine-Related Cancer (2003) 10 193–202 the effect of estrogen antagonists. Several candidate genes ERβcx by stable transfection of each expression plasmid into selected from the contents on the custom-made microarray MCF-7 cells. This constitutive expression of ERβ and ERβcx were also analyzed by real-time RT-PCR and by an immuno- significantly reduced cell growth, cell cycle, and colony for- histochemical technique using breast cancer tissues, in order mation in anchorage-independent situation. ERE activity in to find new predictive factors for the responsiveness to hor- these cells was also strongly diminished compared with par- mone therapy of patients with primary breast cancer. ental MCF-7 cells. Furthermore, endogenous expression of known ERα target genes, such as cathepsin D (Augereau et Regulation of ER␣ gene expression al. 1994), in these cells responded more weakly to estrogen. These observations indicated that both ERβ and ERβcx We previously analyzed promoter usage of ERα gene in inhibit ERα function, resulting in growth inhibition of ERα- human breast cancer tissues, and found that promoter B, one positive breast cancer cells. However, functional differences of the distal promoters,