Endocrine-Related Cancer (2003) 10 193–202

International Congress on Hormonal Steroids and Hormones and Cancer The expression and function of estrogen ␣ and ␤ in human 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 α (ERα) is frequently observed in the early stage of breast cancer. We previously reported that the specific promoter of the ERα 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 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 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 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 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 (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, may be responsible for cancer- between ERβ and ERβcx were observed in the electrophor- specific overexpression of ERα. We next analyzed the pro- etic mobility shift assay and gene expression profiling using moter B region using luciferase reporter plasmids containing microarray. Although nuclear extracts prepared from ERβ- several deleted fragments of this region and gel mobility shift expressing MCF-7 cells formed a DNA–protein complex assay. These experiments identified an important cis-element despite different patterns from parental MCF-7 cells, ERβcx in the downstream region of the transcription initiation site, did not show any clear complexes in this assay. We further which contains the nucleotide sequence CTGGAAAG. This analyzed expression profiles of estrogen-responsive genes in element was capable of transactivating a heterogeneous these cells using our custom-made cDNA microarray as SV40 promoter in MCF-7 cells, confirming that the element described in the next section. The estrogen-responsive is a transcriptional enhancer. The trans-acting factor binding expression profile of the genes in the ERβcx-expressing cells to the element (named ERBF-1) was exclusively expressed was clearly distinct from the ERβ-expressing cells. These in cells expressing ERα mRNA transcribed from promoter B. results suggest that ERβ and ERβcx attenuate ERα function Furthermore, we also attempted to elucidate the mecha- through distinct mechanisms as shown in Fig. 3. We reported nisms of suppression of ERα gene expression in advanced very recently that ERβcx expression may affect the clinical breast cancer. In the development of breast cancer, the loss of outcome of breast cancer, such as the response to tamoxifen ERα gene expression is one of the most important steps in (Saji et al. 2002). acquiring hormone resistance, although the mechanisms are poorly understood. We assessed the effects of methylation in Expression profiling of α the promoter region of ER using breast cancer cell lines and estrogen-responsive genes tissues. The methylation status of promoter B as well as that of promoter A was inversely associated with ERα gene expres- To understand the estrogen-signaling pathway in breast sion. In vitro methylation of ERα directly decreased the tran- cancer cells and to find the molecular markers which reflect scription of the ERα gene in a reporter assay. Demethylating the physiological status of ER-positive breast tumors for treatment induced transcription of ERα from promoter B in clinical application, such as the diagnosis of estrogen- and ZR-75-1 cells which, by their nature, show no transcription anti-estrogen responsiveness of mammary tumors, we ana- from promoter B despite weakERBF-1 expression. On the lyzed the estrogen-responsive gene expression profiles in contrary, ERα-negative cells, such as MDA-MB-231 and human MCF-7 breast cancer cells using a human UniG- BT-20 cells, which lackERBF-1, did not show any induced EMTM V 2.0 microarray system (IncyteGenomics, CA, expression with demethylation. Moreover, ZR-75-1 cells USA) consisting of 9128 human cDNA clones covering 8502 showed ERα promoter activity equal to that of MCF-7 cells unique gene/EST clusters. After MCF-7 cells were cultured when luciferase reporter plasmid was transiently transfected. in estrogen-starved medium for 5 days, the cells were treated These observations indicated that the methylation of promoters with 10 nM 17β-estradiol, which is considered to be a satu- can directly regulate ERα gene expression, and loss of critical rating estrogen-rich condition for the MCF-7 cells but transcription factors such as ERBF-1 may also be involved in physiologically relevant and also a possible condition in the negative regulation of ERα gene expression. As summar- mammary glands. From a total of 9128 clones, the data for ized in Fig. 2, down-regulation of ERα gene expression could 1846 clones were cut off because of low signal intensities as be controlled by two distinct mechanisms: DNA methylation defined by the manufacturer. Among the remaining 7282 of the promoter region or loss of transcriptional activators. clones, 181 genes showed differential expression ratios equal to or more than 2.0, and 105 genes showed differential Function of ER␤ in breast cancer cells expression ratios equal to or less than 0.5; the remaining 96% of the genes revealed no significant differences in their In order to assess the function of ERβ in ERα-positive breast expression levels. In a total of 286 genes which proved to be cancer cells, we established cell lines expressing ERβ and potentially estrogen-responsive genes by this analysis, there www.endocrinology.org Downloaded from Bioscientifica.com at 09/26/2021195 04:32:51AM via free access Hayashi et al.: ERα and β in breast cancer

transcriptional activators ◊ (ex. ERBF-1) ◊ repression (1) methylated promoter transcriptional activators expression HDAC1 (ex. ERBF-1) unmethylated promoter mSin3A ◊ MeCP2 HDAC2 (2) loss of trancriptional activators ◊ repression ◊ ◊ histone deacetylation ◊

◊ repression unmethylated promoter chromatin remodeling

Figure 2 Proposed model of silencing ERα gene expression in breast cancer cells. HDAC, histone deacetylase.

A AF-1 DBD Hinge LBD/AF-2 ERβ A/B CC D EE F

βcx A/B CC D EE X splicing variants in the C-terminal region B heterodimer ERα ERβ > ERE ERE > ERE

βcx ERβ Target genes Clinical outcome PgR etc response to tamoxifen

Figure 3 (A) Structure of wild-type ERβ and ERβcx and (B) how they modulate the transcription activity of ERα through the distinct mechanisms. AF-1, activation function 1; DBD, DNA binding domain; LBD, ligand binding domain; PgR, .

196 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/26/2021 04:32:51AM via free access Endocrine-Related Cancer (2003) 10 193–202

Table 1 Expression ratios of 148 genes loaded on the custom-made cDNA microarray Accession no. Gene name Group) Ratio of expression at 72 hrsa Group A: early-responsive estrogen-induced genes M80244 solute carrier family 7, member 5 8.34 AJ011972 histone deacetylase 6 4.75 AB018010 antigen identified by monoclonal 4F2, TRA1.10, TROP4, and T43 4.70 X84373 interacting protein 1 3.78 M62403 insulin-like growth factor-building protein 4 3.58 X05030 trefoil factor 1 3.34 U44427 tumor protein D52-like 1 2.85 D10495 protein kinase C, delta 2.78 AB028974 KIAA1051 protein 2.71 X16706 FOS-like antigen 2 2.20 D13643 KIAA0018 2.02 M63138 cathepsin D 1.88 L06237 microtubule-associated protein 1B 1.84

Group B: late-responsive oestrogen-induced genes AF012281 PDZ domain containing 1 6.64 AA587912 ESTs, Highly similar to phosphoserine aminotransferase 5.54 U72066 retinoblastoma-binding protein 8 5.33 N3555 EST 4.94 L02785 down-regulated in adenoma 4.16 X16396 methylene tetrahydrofolate dehydrogenase (NAD+ dependent), methenyltetrahydrofolate cyclohydrolase 4.13 X62570 tryptophanyl-tRNA synthetase 4.01 U29343 hyaluronan-mediated motility receptor (RHAMM) 3.76 AI767533 EST 3.70 AA430241 pituitary tumor-transforming 1 3.53 AI660571 argininosuccinate synthetase 3.53 D30658 glycyl-tRNA synthetase 3.42 X62585 EST 3.38 X72875 B-factor, properdin 3.24 AB018265 unc-51 (C. elegans)-like kinase 1 3.21 L35946 asparagine synthetase 3.18 X92720 phosphoenolpyruvate carboxykinase 2 (mitochondrial) 3.02 AF039022 exportin, tNRA (nuclear export receptor for tRNAs) 2.89 U66838 cyclin A1 2.87 AI880413 Incyte EST 2.79 AA629308 ESTs, Highly similar to BimL 2.76 AI151190 S100 calcium-binding protein B 2.75 AI878886 heat shock 70kD protein 5 (glucose-regulated protein, 78kD) 2.65 X89773 interferon-stimulated gene (20kD) 2.61 D90070 phorbol-12-myristate-13-acetate-induced protein 1 2.56 N39944 activating 3 2.53 AF050110 TGFB inducible early growth response 2.52 AB011123 KIAA0551 2.51 X74837 mannosidase, alpha, class 1A, member 1 2.47 U23143 serine hydroxymethyltransferase 2 (mitochondrial) 2.47 AB012664 stanniocalcin 2 2.44 M37400 glutamic-oxaloacetic transaminase 1, soluble (aspartate aminotransferase 1) 2.42 AA216685 prostate differentiation factor 2.32 M90516 glutamine-fructose-6-phosphate transaminase 1 2.26 L14595 solute carrier family 1 (glutamate/neutral amino acid transporter), member 4 2.24 AB005047 SH3-domain-binding protein 5 (BTK-associated) 2.24 AA102267 ferritin, heavy polypeptide 1 2.14 D42073 reticulocalbin 1, EF-hand calcium-binding domain 2.13 AI1185199 EST 2.07 U82984 ESTs, Weakly similar to oligophrenin-1 like protein 2.04 N28312 HS1 binding protein 2.03 AB011159 KIAA0587 2.03

Group C: oestrogen-repressed genes X76220 mal, T-cell differentiation protein − 2.00 U59325 cadherin 18 − 2.04 AI700706 general transcription factor II, i, pseudogene 1 − 2.21 D87993 paired basic amino acid cleaving system 4 − 2.34 U29091 selenium binding protein 1 − 2.37 AJ002308 synaptogyrin 2 − 2.48 U96136 catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein) − 2.60 AB002305 KIAA0307 gene product − 2.65 X13425 membrane component, 1, surface marker 1 (40kD glycoprotein, identified by monoclonal GA733) − 2.65 AC002984 calpain, small polypeptide − 2.69 M19922 fructose-bisphosphatase 1 − 2.76 M59828 heat shock 70kD protein 1 − 2.94 X69433 isocitrate dehydrogenase 2 (NADP+ ), mitochondrial − 3.23 X69550 Rho GDP dissociation inhibitor (GDI) alpha − 3.38 AA374325 insulin-like growth factor binding protein 5 − 3.46 U03877 EGF-containing fibulin-like extracellular matrix protein 1 − 3.94 U97276 quiescin Q6 − 4.13 X56832 3, (beta, muscle) − 5.24 X51956 enolase 2, (gamma, neuronal) − 5.42 AA482422 promoter-binding protein 1 − 5.79

www.endocrinology.org Downloaded from Bioscientifica.com at 09/26/2021197 04:32:51AM via free access Hayashi et al.: ERα and β in breast cancer

Table 1 Continued Accession no. Gene name (Group) Ratio of expression at 72 hrsa U30246 solute carrier family 12 (sodium/potassium/chloride transporters), member 2 − 5.84 L27560 Human insulin-like growth factor binding protein 5 (IGFBP5) − 8.42

Others: genes without significant response to estrogen M84755 Neuropeptide Y receptor Y1 1.97 D28473 Isoleucine-tRNA synthetase 1.95 AI949781 ESTs, Weakly similar to phosphoprotein 1.95 AF105826 Solute carrier family 1 (neutral amino acid transporter), member 5 1.94 X15187 Tumor rejection antigen (gp96) 1 1.91 AI492976 ESTs, Moderately similar to KIAA0400 1.91 AA557306 CCAAT/enhancer binding protein (C/EBP), beta 1.89 U08316 Ribosomal protein S6 kinase, 90kD, polypeptide 3 1.86 AI332415 EST 1.85 U89436 Tyrosyl-tRNA synthetase 1.85 L20815 Corneodesmosin 1.84 AI261366 DKFZP566G223 protein 1.81 AA481712 Protein geranylgeranyltransferase type I, beta subunit 1.76 AI188401 ESTs, highly similar to CGI-82 protein 1.75 AI683760 Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3B 1.74 U21858 TATA box-binding protein (TBP)-associated factor, RNA polymerase II, G, 32kD 1.74 X64I16 Poliovirus receptor 1.71 AI088306 FOS-like antigen 2 1.65 AI598150 v-jun avian virus 17 homolog 1.60 J05068 Transcobalamin I(vitamin B12-binding protein, R binder family) 1.59 Z48950 H3 histone, family 3B (H3 3B) 1.59 X06233 S100 calcium-binding protein A9 (calgranulin B) 1.59 AF065388 Tetraspan 1 1.56 T73188 Aldo-keto reductase family 1, member C4 (chlorodecone reductase; 3-alpha-hydroxysteroid dehydrogenase, type I; dihydrodiol dehydrogenase 4) 1.54 D32050 Alanyl-tRNA synthetase 1.53 AF022109 CDC6 (cell division cycle 6, S. cerevisiae) homolog 1.52 AA975298 EST 1.52 AA484893 Matrix Gla protein 1.47 AI342303 Solute carrier family 29 (nucleoside transporters), member 2 1.36 Y18448 Bassoon (presynaptic cytomatrix protein) 1.33 AA143530 stearoyl-CoA desaturase (delta 9-desaturase) 1.31 AA682502 EST 1.29 AF055008 granulin 1.27 AL022726 inhibitor of DNA binding 4, dominant negative helix-loop-helix protein 1.23 Z80998 solute carrier family 5 (sodium/glucose cotransporter), member 1 1.22 X55122 GATA-binding protein 3 1.21 X63741 early growth response 3 1.21 L35546 glutamate-cysteine (gamma-glutamylcysteine synthetase), regulatory (30.8kD) 1.18 D21205 zinc finger protein 147 (oestrogen-responsive finger protein) 1.17 AA994925 tumor necrosis factor receptor superfamily, member 7 1.08 AI239815 general transcription factor IIH, polypeptide 2 (44kD subunit) 1.07 Z46973 phosphoinositide-3-kinase, class 3 1.07 L42611 keratin 6B 1.05 AI679881 thrombospondin 3 1.02 X15393 motilin 1.02 U07361 sorbitol dehydrogenase − 1.02 X74929 keratin 8 − 1.11 X00947 alpha-1-antichymotrypsin − 1.14 AL046741 chromobox homolog 1 (Drosophila HP1 beta) − 1.19 U79302 Human clone 23855 mRNA, partial cds − 1.20 AA173870 nucleophosmin (nuclear phosphoprotein B23, numatrin) − 1.22 U78525 eukaryotic translation initiation factor 3, subunit 8 (eta, 116kD) − 1.28 D21163 U5 snRNP-specific protein, 116kD) − 1.31 M30704 amphiregulin (schwannoma-derived growth factor) − 1.38 X00351 actin, beta − 1.43 L39211 carnitine palmitoyltransferase I, liver − 1.44 X14723 clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, -repressed prostate message 2, apolipoprotein J) − 1.45 AI815757 ribosomal protein L35 − 1.45 D21064 KIAA0123 − 1.47 Y17977 fucosyltransferase 8 (alpha (1,6) fucosyltransferase) − 1.49 AL036958 KIAA0058 − 1.57 AF0544996 Homo sapiens clone 23783 mRNA − 1.58 AC004030 Homo sapiens DNA from chromosome 19, cosmid F21856 − 1.61 U83115 absent in 1 − 1.62 AF014807 CDP-diacylglycerol – inositol 3-phosphatidyltransferase (phosphatidylinositol synthase) − 1.66 U87939 2, mitochondrial − 1.71 D83780 KIAA0196 − 1.76 X79568 protein tyrosine phosphatase, non-receptor type 18 (brain-derived) − 1.83 X03635 estrogen receptor 1 − 1.84 L25081 ras homolog gene family, member C − 1.93 J03075 protein kinase C substrate 80K-H − 1.96 a ‘Ratio of expression at 72 hrs’ means the ratio of expression levels for each gene in MCF-7 cells treated with (E2+) and without (E2−) 17β-estradiol for 72 hrs, which examined by custom-made microarray analysis. For E2 -upregulated or E2 -downregulated genes, the values are shown as the ratios E2+/E2 –or–(E2-/E2+) respectively.

198 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/26/2021 04:32:51AM via free access Endocrine-Related Cancer (2003) 10 193–202 were some genes which had previously been reported to be removing the genes which showed relatively low ratios of induced by estrogen such as pS2 (Brown et al. 1984), estrogen-responsive expression (near or equal to 2.0 or PDZK1 (PDZ domain-containing protein) (Ghosh et al. − 2.0) and which were inferred to have little relation to the 2000), and insulin-like growth factor-binding protein nature of breast cancer according to the background infor- (IGFBP)-4 (Qin et al. 1999), indicating the reliability of this mation on the genes. Using this customized microarray analysis. prototype, we analyzed the time-course of estrogen response From the 286 potentially estrogen-responsive genes men- of the genes in MCF-7 cells. The estrogen-starved MCF-7 tioned above, a total of 138 genes were selected for pro- cells were treated with ethanol (E2 − ) (Cy5-labelled) or 10 duction of a prototype of the custom-made microarray, nM 17β-estradiol (E2 + ) for 6, 12, 24 or 72 h (Cy3-labelled),

A 9

-) trefoil factor 1 (pS2) 2 8 7 IGF-binding protein 4 + / E 2 6 KIAA1051 protein 5 solute carrier family 7, member 5 4 histone deacetylase 6 3 2 cathepsin D

Fold Expression (E 1 tumor protein D52-like 1 0 0 122436486072 Time after estrogen addition (h)

retinoblastoma-binding protein 8 B 8

-) asparagine synthetase 2 7 EST + / E 6 2 5 ESTs, Highly similar to phosphoserine aminotransferase 4 cyclin A1 3 down-regulated in adenoma 2 pituitary tumor-transforming 1

Fold Expression (E 1 tryptophanyl-tRNA synthetase 0 0 122436486072 Time after estrogen addition (h) C 0 IGF-binding protein 5 +)

2 -1 MYC promoter-binding protein 1 E

-/ enolase 2 (gamma, neuronal)

2 -2 -3 KIAA0307 -4 quiescin Q6 solute carrier family 12, member 2 -5 selenium binding protein 1 -6

Fold Expression (-E EGF-containing fibulin-like -7 extracellular matrix protein 1 0 122436486072 Time after estrogen addition (h)

Figure 4 Expression profiles of several genes in MCF-7 cells at different times after estrogen stimulation analyzed by customized microarrays. Several representative genes in each group were selected and the time-courses of the ratios of expression levels between the cells treated with E2 + and E2 − are plotted. (A), (B) and (C) show the expression patterns of early-responsive estrogen-induced genes, late-responsive estrogen-induced genes and estrogen-repressed genes respectively. www.endocrinology.org Downloaded from Bioscientifica.com at 09/26/2021199 04:32:51AM via free access Hayashi et al.: ERα and β in breast cancer and mRNA isolated from each culture of the cells was used Figure 4 shows the time-course profile of estrogen- for the microarray analysis. The mRNA was also isolated responsive expression of several genes selected from groups from the estrogen-starved cells but without any treatment A, B and C. As expected, pS2, IGFBP-4 and cathepsin D, (shown as 0 h). On the customized microarray, cDNA frag- which were reported to be the target genes of ERα, were ments for each gene were spotted in two blocks, yielding a found in the group of early-responsive estrogen-induced pair of data sets of expression ratios from one hybridization. genes (Fig. 4A). IGFBP-5, which was reported to be down- In the case of estradiol treatment for 0 h (without estradiol regulated by estrogen (Huynh et al. 1996), was found in the treatment), Cy3- and Cy5-labeled cDNA were produced from group of estrogen-repressed genes (Fig. 4C). Among these the same preparation of mRNA and hybridized together into genes listed in Table 1, we performed Northern blot analysis one microarray glass slide, for the purpose of examining the for several of them to confirm the results from microarray level of technical variation in fluorescent labeling and/or analysis, and all the examined genes exhibited similar hybridization. As a result, all the Cy3/Cy5 ratios for the expression patterns in Northern analysis to that from microar- genes were between 0.5 and 2.0. Hierarchical clustering ray analysis (data not shown). clearly highlighted the gene clusters of estrogen-induced and We have developed a second version of the custom-made estrogen-repressed genes, and made it possible to subdivide estrogen-responsive array (estrogen-responsive gene (ERG) the cluster of estrogen-induced genes into two subgroups; chip in Fig. 5), and are currently applying it to various one group containing the genes which showed expression studies such as basic research for estrogen signaling and ratios above 2.0 after 12 h of estrogen treatment clinical application for the diagnosis of estrogen-dependent (early-responsive estrogen-induced genes, group A) and the cancer, as summarized in Fig. 5. other group containing the genes which showed significant induction after 24 h or, in most cases, 72 h of estrogen treat- Identification of predictive factors for ment (late-responsive estrogen-induced genes, group B). On endocrine therapy the other hand, the cluster of estrogen-repressed genes (group C) did not show any apparent subgroups. The expression Based on the results obtained from the microarray analysis, ratios after estradiol treatment for 72 h of all the 148 genes we selected 11 genes as candidate factors for predicting the loaded on the custom-made microarray are listed in Table 1, efficacy of endocrine therapy. We assessed their expression being divided into four groups including groups A, B and C. levels in breast cancer tissues by real-time RT-PCR, and the

Microarray analysis of 10000 genes

Identification of estrogen regulated genes (Estrogen Responsive Genes)

Production of custom-made cDNA microarray

ERG chip (200 genes)

Basic research for estrogen Diagnosis of hormone- Screening of novel signaling pathway dependent cancers prognostic factors

•Screening of ER-target genes •Prediction of hormone therapy •Functional analyses of ERα, ERβ •Classification of patients for individualized therapy •Evaluation of SERMs •Monitoring of hormone therapy •Mechanisms of anti-hormone resistance

Figure 5 Development of the custom-made estrogen-responsive cDNA microarray and its application to basic and clinical studies. SERMS; selective estrogen receptor modulators.

200 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/26/2021 04:32:51AM via free access Endocrine-Related Cancer (2003) 10 193–202

Gene A Gene B

1 1 Low (n=47) Low (n=15) .8 .8

.6 .6 High (n=50) High (n=18) .4 .4

.2 .2 p=0.02 (Logrank (Mantel-Cox) test) p=0.04 (Logrank (Mantel-Cox) test) Disease-free survival rate p=0.01 (Breslow-Gehan-Wilcoxon test) Disease-free survival rate p=0.06 (Breslow-Gehan-Wilcoxon test) 0 0 0 1000 2000 3000 4000 0 1000 2000 3000 4000 Days Days

Figure 6 Kaplan–Meier analysis of disease-free survival rates of patients who had tamoxifen adjuvant therapy and the expression status of selected candidate genes, Gene A and Gene B. result was analyzed by cluster analysis. ER-positive patients useful for predicting the efficacy of endocrine therapy for were clearly divided into two subgroups that were expected individual patients with breast cancer. In fact, along with to represent different estrogen responsiveness. The same sub- recent progress, novel endocrine therapies, including aromat- groups were almost reproducible with a few representative ase inhibitors, a new accurate prediction method for these genes in this 11 gene subset. We therefore examined the treatments is urgently required. Finally, this study has sug- expression of these genes by immunohistochemical analysis gested that new predictive factors could be identified in in 65 patients treated with adjuvant tamoxifen therapy, in estrogen-responsive genes that distinguish responsive or non- order to assess the impact on the prediction of the effect of responsive patients to tamoxifen treatment in an ER-positive this endocrine therapy. As shown in Fig. 6, positive staining population. Immunohistochemical evaluation using these fac- of gene A as well as gene B significantly correlated with tors may be a more practical method than cDNA microarray. lower disease-free survival rate. Furthermore, combined Although further study will be needed, these approaches assessment of expression of these genes improved the predic- could provide not only new clues for the elucidation of tive accuracy for tamoxifen response. mechanisms of estrogen-dependent carcinogenesis and the development of breast cancer, but also clinical benefits to Conclusion and perspective patients by assessment of individual responses to endocrine therapy. These studies of the expression and function of ERs, especially from the aspect of cancer-specific phenomena, are Acknowledgements very important for improving new strategies of prevention, diagnosis, and therapy of estrogen-dependent cancers. For We thankMs AkiyoYamashita for her excellent technical instance, elucidation of the mechanism of the regulation of assistance, Dr Masakazu Toi, Dr Ryoichi Kiyama and Dr Kei ERα gene expression may provide a new idea to prevent or Nakachi for their valuable suggestions and advice. This study arrest the development of breast cancer, and ERβcx expres- was supported in part by Grants-in-Aid from the Ministry of sion status may assist in the eligibility of therapeutic options. Education, Culture, Sports, Science and Technology of Japan On the other hand, cDNA microarray technique is another for Cancer Research, from the Ministry of Health, Labor and very promising method for these studies. We are currently Welfare of Japan for Scientific Research Expenses for Health applying our custom-made cDNA microarray to extensive and Welfare Programs and the Foundation for the Promotion studies on estrogen signaling in terms of both basic and clini- of Cancer Research and for Second-Term Comprehensive cal aspects. This down-sized microarray may be especially 10-Year Strategy for Cancer Control. www.endocrinology.org Downloaded from Bioscientifica.com at 09/26/2021201 04:32:51AM via free access Hayashi et al.: ERα and β in breast cancer

References Omoto Y, Kobayashi Y, Nishida K, Tsuchiya E, Eguchi H, Nakagawa K, Ishikawa Y, Yamori T, Iwase H, Fujii Y, Warner Augereau P, Miralles F, Cavaille`s V, Gaudelet C, Parker M & M, Gustafsson J-Å & Hayashi S-I 2001 Expression, function and Rochefort H 1994 Characterization of the proximal clinical implications of estrogen receptor β in human lung estrogen-responsive element of human cathepsin D gene. cancer. Biochemical and Biophysical Research Communications Molecular Endocrinology 8 693–703. 285 340–347. Brown AMC, Jeltsch JM, Roberts M & Chambon P 1984 Omoto Y, Kobayashi S, Inoue S, Ogawa S, Toyama T, Yamashita Activation of pS2 gene transcription is a primary response to H, Muramatsu M, Gustafsson J-Å & Iwase H 2002 Evaluation estrogen in the human breast cancer cell line MCF-7. PNAS 81 of oestrogen receptor β wild-type and variant protein expression, 6344–6348. and relationship with clinicopathological factors in breast Finlin BS, Gau C-L, Murphy GA, Shao H, Kimel T, Seitz RS, cancers. European Journal of Cancer 38 380–386. Chiu Y-F, Botstein D, Brown PO, Der CJ, Tamanoi F, Andres Qin C, Singh P & Safe S 1999 Transcriptional activation of DA & Perou CM 2001 RERG is a novel ras-related, insulin-like growth factor-binding protein-4 by 17β-estradiol in estrogen-regulated and growth-inhibitory gene in breast cancer. MCF-7 cells: role of estrogen receptor–Sp1 complexes. Journal of Biological Chemistry 276 42259–42267. Endocrinology 140 2501–2508. Ghosh MG, Thompson DA & Weigel RJ 2000 PDZK1 and Saji S, Okumura N, Eguchi H, Nakashima S, Suzuki A, Toi M, GREB1 are estrogen-regulated genes expressed in Nozawa Y, Saji S & Hayashi S-I 2001 MDM2 enhances the hormone-responsive breast cancer. Cancer Research 60 6367– function of estrogen receptor α in human breast cancer cells. 6375. Biochemical and Biophysical Research Communications 281 Hayashi S-I, Imai K, Suga K, Kurihara T, Higashi Y & Nakachi K 259–265. 1997a Two promoters in expression of estrogen receptor Saji S, Omoto Y, Shimizu C, Warner M, Hayashi Y, Horiguchi S, messenger RNA in human breast cancer. Carcinogenesis 18 Watanabe T, Hayashi S-I, Gustafsson J-Å & Toi M 2002 459–464. Expression of estrogen receptor (ER) βcx protein in Hayashi S-I, Hajiro-Nakanishi K, Makino Y, Eguchi H, Yodoi J & ERα-positive breast cancer; specific correlation with Tanaka H 1997b Functional modulation of estrogen receptor by progesterone receptor. Cancer Research 62 4849–4853. redox state with reference to thioredoxin as a mediator. Nucleic Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Acids Research 25 4035–4040. Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Huynh H, Yang X-F & PollakM 1996 A role for insulin-like Quist H, Matese JC, Brown PO, Botstein D, Lønning PE & growth factor binding protein 5 in the antiproliferative action of B¿rresen-Dale A-L 2001 Gene expression patterns of breast the antiestrogen ICI 182780. Cell Growth and Differentiation 7 carcinomas distinguish tumor subclasses with clinical 1501–1506. PNAS 98 Inoue A, Yoshida N, Omoto Y, Oguchi S, Yamori T, Kiyama R & implications. 10869–10874. Hayashi S-I 2002 Development of cDNA microarray for Soulez M & Parker MG 2001 Identification of novel oestrogen expression profiling of estrogen-responsive genes. Journal of receptor target genes in human ZR75-1 breast cancer cells by Molecular Endocrinology 29 175–192. expression profiling. Journal of Molecular Endocrinology 27 Kuiper GGJM, Enmark E, Pelto-Hukko M, Nilsson S & 259–274. Gustafsson J-A 1996 Cloning of a novel estrogen receptor Tanimoto K, Eguchi H, Yoshida T, Hajiro-Nakanishi K & Hayashi α expressed in prostate and ovary. PNAS 93 5925–5930. S-I 1999 Regulation of estrogen receptor gene mediated by Leygue E, Dotzlaw H, Watson PH & Murphy LC 1999 Expression promoter B responsible for its enhanced expression in human of estrogen receptor β1, β2, and β5 messenger RNAs in human breast cancer. Nucleic Acids Research 27 903–909. breast tissue. Cancer Research 59 1175–1179. Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, Matsuyama S, Ohkura Y, Eguchi H, Kobayashi Y, Akagi K, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Uchida K, Nakachi K, Gustafsson J-Å & Hayashi S-I 2002 Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards Estrogen receptor β (ERβ) is expressed in human stomach R & Friend SH 2002 Gene expression profiling predicts clinical adenocarcinomas. Journal of Cancer Research and Clinical outcome of breast cancer. Nature 415 530–536. Oncology 128 319–324. Yoshida T, Eguchi H, Nakachi K, Tanimoto K, Higashi Y, Ogawa S, Inoue S, Watanabe T, Orimo A, Hsoi T, Ouchi Y & Suemasu K, Iino Y, Morishita Y & Hayashi S-I 2000 Distinct Muramatou M 1998 Molecular cloning and characterisation at mechanisms of loss of estrogen receptor α gene expression in human estrogen receptor BCX: a potential inhibitor of estrogen human breast cancer: methylation of the gene and alteration of action in human. Nucleic Acids Research 26 3502–3512. trans-acting factors. Carcinogenesis 21 2193–2201.

202 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/26/2021 04:32:51AM via free access