Oct 17, 1996 - The human estrogen receptor (ER) gene has recently been predictor of .... that the enhanced ER protein expression in breast tumors correlates ...
Carcinogenesis vol.18 no.3 pp.459–464, 1997
Two promoters in expression of estrogen receptor messenger RNA in human breast cancer
Shin-ichi Hayashi1,5, Kazue Imai2, Kenji Suga2, Terumasa Kurihara3,4, Yasuhiro Higashi3 and Kei Nakachi2 1Department
of Biochemistry and 2Department of Epidemiology, Saitama Cancer Center Research Institute, and 3Department of Breast Surgery, Saitama Cancer Center Hospital, 818 Komuro, Ina, Saitama 362, Japan 4Present
address: Department of Surgery, National Nishi-Gunma Hospital, 2854 Kanai, Shibukawa, Gunma 377, Japan 5To
whom correspondence should be addressed
The human estrogen receptor (ER) gene has recently been shown to transcribe two types of mRNA originating from two distinct promoters in mammary tumor cell lines, which encode the same protein. However, use of the two promoters has not been addressed in human breast cancer, which reveals a heterogeneity in terms of ER expression status and clinical characteristics. In this report, we investigated which promoter is responsible for the expression of ER in human mammary tumors by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis for discriminatory detection of the two transcripts in mammary tissues obtained from patients with breast cancer. First, the use of distinct promoters was confirmed in several mammary tumor cell lines by the present RT-PCR method. Secondly, expression levels of total ER mRNA and two types of mRNAs from the different promoters were analysed in tumor, surrounding tissue and normal tissue obtained from 12 patients with breast cancer, which showed various levels of ER protein. In tumors, levels of total ER mRNA and the mRNA transcribed from a distal promoter showed remarkable correlation to the ER protein levels with correlation coefficients 0.946 (P < 0.001) and 0.746 (P < 0.005), respectively. In contrast, mRNA from a proximal promoter showed no correlation to the ER protein levels. Our results indicate that the enhancement of the ER mRNA expression from the distal promoter plays an essential role in the mechanisms of overexpressing ER protein in human mammary tumors, implying that a tumor-specific regulation of ER expression involved use of the distal promoter.
As a mediator of estrogen effect on the target cells, estrogen receptor (ER*) plays an important role in regulating growth and differentiation of normal breast epithelium (3): ER regulates the transcription of various genes as a transcription factor, which binds to estrogen response elements upstream of the target genes (4,5). In breast carcinogenesis, the expression of ER is closely associated with the cancer biology, especially the development of tumor, i.e. breast carcinomas which lack ER expression often reveal more aggressive phenotypes (6–8). Moreover, the expression of ER in tumor tissues is a good predictor of prognosis in endocrine treatment (9), which aims to inhibit the mitogenic stimulus produced by the ER bound to estrogen. A number of studies have been carried out to find the mechanisms responsible for the lack of ER expression, and they have reported many splice variants of ER mRNA together with insertion, deletion and point mutations of the ER gene (10–16). However, mutations can account for only a small portion of ER-negative phenotypes, and splice variants cannot account for those phenotypes since they are often expressed in concomitance to the normal transcript (17–20). Studying the mechanisms of regulating transcription of the ER gene may provide a new insight into understanding of breast carcinogenesis, although little is known on the transcriptional regulation of this gene. ER belongs to a family of ligand-inducible nuclear receptors that have steroid and thyroid hormones as known ligands. The human ER gene is located on chromosome 6q25.1 spanning . 140 kb, and it contains eight exons and eight introns (21,22). After cloning cDNA and partial genomic DNA of the ER gene (23), several laboratories have attempted to identify and analyse the upstream region of this gene, revealing that the transcription
Introduction Some human cancers have been studied from the aspect of host-environment interactions to characterize individual cancer incidence such as genetic susceptibility in lung cancer (1) and in breast and ovarian cancers (2). Furthermore, interactions between tumors and their micro-environment are of significance in the development and progression of individual tumors, e.g. hormonal environment, and expression of hormone receptors in breast and prostate carcinogenesis. *Abbreviations: ER, estrogen receptor; GAPDH, glyceraldehyde phosphate dehydrogenase; RT-PCR, reverse transcription-polymerase chain reaction; EIA, enzyme immuno-assay. © Oxford University Press
Fig. 1. Genomic organization map of the human ER gene at the 59 end. Arrowheads indicate the positions of the primers used in RT-PCR, i.e. primers 10 and 9 for detection of total ER mRNA, primers 13 and 9 for transcripts from promoter A, and primers 1 and 8 for those from promoter B. Bent arrows indicate the positions of the two transcription start sites from distinct promoters of the human ER gene. The transcripts from promoter A and promoter B are tentatively named as type A mRNA and type B mRNA, respectively.
S.-I.Hayashi et al.
Fig. 2. Transcripts from the two promoters in the ER receptor gene, detected in MCF-7, ZR-75–1 and MBA-MD-231 cells, by RT-PCR. MCF-7 and ZR-75–1 are ER-positive cell lines, while MDA-MB-231 is ER-negative. Total RNAs from each cell were transcribed into cDNA using reverse transcriptase and random hexamers as primers. These cDNAs were applied to subsequent PCR reaction as described in Materials and methods. The PCR products were subjected to 2% agarose gel electrophoresis, and stained with ethidium bromide (A), or autoradiographed in 5% polyacrylamide-gel electrophoresis (B). Total ER, type A and type B mRNA were detected in MCF-7 (lane 1); total ER and type A mRNA in ZR-75–1 (lane 2); none of these mRNAs in MDA-MB-231 (lane 3). Total ER; the PCR product obtained from primers 10 and 9, type A; that from primers 13 and 9, type B; that from primers 1 and 8. Marker; commercial molecular weight marker (DNA ladder, BRL).
Fig. 3. ER protein levels among 50 patients with breast cancer. Enzyme immuno-assays for tumor samples were performed with ER-EIA kits, and ,10 fmol/mg protein was considered negative. Triangles indicate the twelve patients studied by the RT-PCR analysis, who were randomly chosen from patient groups with ER-negative tumors, ER-positive tumors at low levels and those at high levels.
of this gene occurs from two different promoters (24–26). Transcription from the two promoters has been characterized on various cell lines including mammary tumor cell lines (27,28). A promoter immediately upstream of the coding region has predominantly been used in normal human mammary epithelium and also in ER-positive mammary tumor cell lines, while the message from the other distal upstream promoter has also been detected in mammary tumor cell lines at different levels of expression. Now required is a quantitative analysis 460
of the transcription originating from the two different promoters on tumor tissues obtained from patients with breast cancer, who have different status of ER expression and clinical characteristics. To elucidate which promoter is responsible for the expression of ER in human mammary tumor tissues, we carried out a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis on RNAs prepared from tumor, its surrounding tissue, and normal mammary tissue of 12 breast cancer patients. In this report, we showed that the enhanced ER protein expression in breast tumors correlates to the mRNA expression from the distal upstream promoter, not that from the promoter proximal to the coding region, indicating that the transcription from the distal upstream promoter plays an essential role in the mechanisms of overexpressing ER protein in human breast carcinogenesis. Materials and methods Cells and culture Human mammary tumor cell lines (MCF-7, ZR-75–1, MDA-MB-231) were cultured in suspension in RPMI1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 5 units/ml penicillin, and 5 µg/ml streptomycin (Sigma) at 37°C in a humidified atmosphere of 5% CO2 in air. Tissue samples Samples of human primary breast carcinoma, surrounding tissue and normal mammary tissue from 50 female patients were obtained from Saitama Cancer Center Hospital, Japan. Here, ‘surrounding’ means that surgeons dissected non-cancerous tissue adjacent to tumor according to their macroscopic judgement. Tumor samples obtained in surgery were immediately dissected to remove residual normal tissue and then subjected to RNA preparation. Preparation of RNA and semi-quantitative RT-PCR Total RNAs were prepared from cultured cells (1–53106 cells) and ~0.1 g human mammary tissue according to the method of Chomczynski and Sacchi
mRNA expression in breast cancer
Fig. 4. Typical patterns of ER mRNA expression from the two promoters in human breast tissues. N; normal mammary gland tissue, S; surrounding mammary gland tissue (adjacent to tumor), T; tumor breast tissue. The numbers above lanes indicate the patients with various levels of ER protein, 1; ERnegative, 2; ER-positive at a low levels, 3 and 4; ER-positive at high levels.
(29). Semi-quantitative RT-PCR were carried out using the GeneAmp RNA PCR Kit (Takara Shuzo Co., Ltd., Tokyo) as previously reported (30). Oligonucleotides used in PCR amplification were as follows: hER1, CTC GCA CAT GCG AGC ACA TT; hER8, GCT CGT TCC CTT GGA TCT GA; hER9, GGT ACT GGC CAA TCT TTC TC; hER10, AAC GCG CAG GTC TAC GGT CA; hER13, TAA CCT CGG GCT GTG CTC TT for ER (23); GAP1, ACA TCG CTC AGA CAC CAT GG, and GAP2, GTA GTT GAG GTC AAT GAA GGG for glyceraldehyde phosphate dehydrogenase (GAPDH) (31). The primers were designed to sandwich one intron for specific detection of mRNA. The prepared RNA (1 µg) was reverse transcribed to synthesize cDNA using random hexamers at 42°C and then subjected to PCR amplification with specific primers (0.4 µg) and 3 µCi of [α-32P]dCTP (3000 Ci/mmol) in 50 µl mixtures consisting of 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, and 0.2 mM dNTPs (dATP, dTTP, dGTP, dCTP). PCR comprised 25 cycles for GAPDH using primers GAP1 and GAP2, and 30 cycles for ER using the primers shown in Figure 1, with denaturing at 95°C for 1 min, annealing at 65°C for 1 min and extension at 72°C for 1 min in each cycle using a GeneAmpTM PCR System 9600 (Perkin Elmer Cetus). The PCR products were then subjected to 5% polyacrylamide gel-electrophoresis, and the radioactivity was evaluated by autoradiography with the Fuji Bio-Image Analyser BAS2000 (Fuji Film Co., Ltd., Tokyo). ER protein assay ER enzyme immuno-assays for tumor samples were performed with ER-EIA kits (Abbott). ER values ,10 fmol/mg protein were considered negative.
Results Detection of ER transcripts from two promoters in mammary tumor cell lines Recent studies have shown that the human ER gene is transcribed from different promoters, i.e. the distal promoter (promoter B) is located at 2 kb upstream of the proximal one (promoter A), and the primary transcript originating from promoter B is spliced as shown in Figure 1. This implies that there exist different mechanisms in regulating the transcription of ER expression, since the structures of these promoters were
entirely different from each other. The resultant transcripts from the two promoters differ only at the edge of 59 end in the non-coding region and these two ER mRNAs encode the same protein of 595 amino acids. Northern blotting cannot discriminate the two ER mRNAs since the spliced mRNAs are almost the same size (Figure 1). However, we were able to discriminate the transcripts from the two promoters using a RT-PCR analysis as shown in Figure 1. Primers 1 and 8 were used to amplify the cDNA fragment of 237 bp specific for the transcript from promoter B, whereas primers 13 and 9 amplified the fragment of 601 bp specific for that from promoter A. Primers 10 and 9 were used to amplify the fragment of 300 bp in the coding region for detection of total ER mRNA. The RT-PCR products generated with total RNA from various mammary tumor cell lines were shown in Figure 2. Ethidium bromide stained RT-PCR products in MCF7 cells showed single major bands for total ER mRNA, mRNA from promoter A (type A), and mRNA from promoter B (type B) at the predicted positions (Figure 2A), confirming the specific detection of our RT-PCR. Semi-quantitative RT-PCR has been carried out with RNA from several mammary tumor cell lines, as shown in Figure 2B. The PCR products were detected as single major bands for total ER, type A and type B mRNA in MCF-7, and for total ER and type A mRNA in ZR-75–1 cells, but not detected in MDA-MB-231 cells which are known to be an ER-negative cell line. Linear increase of radio-labeled PCR products with increased amounts of RNA was ascertained as described in a previous report (30). These results indicate that this semi-quantitative RT-PCR method can discriminate relative levels of the ER transcript originating from each of the distinct promoters among the different cell lines at high sensitivity, although the absolute levels or ratios 461
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Table I. Relative expression levels of ER mRNAs among 12 patients with breast cancer Patient No.
ER (fmol/mg protein)
Type A mRNA
Type B mRNA
N S T N S T N S T N S T N S T N S T N S T N S T N S T N S T N S T N S T
15 22 ND 12 4 ND 18 41 17 11 37 20 7 5 111 14 7 113 42 37 112 28 6 168 5 19 181 42 18 126 11 23 264 43 16 315
1.1 1.3 ND 1.1 1.2 ND 1.6 1.2 2.0 4.0 4.8 5.2 ND 1.8 8.0 1.3 1.5 70 1.9 1.5 32 ND 1.6 1.8 4.3 2.2 34 3.2 1.8 10 3.5 3.3 4.0 2.1 1.8 6.2
ND 5 ND 4 ND ND 8 18 4 50 4 10 4 ND 107 6 9 196 5 5 210 9 6 42 15 21 352 3 ND 162 ND 7 369 12 16 285
aPathological classification by UICC. bMessenger RNA expression of total ER,
types A and B in tumor, surrounding and normal tissues from each patient were analysed by a semi-quantitative RTPCR method, and presented as relative ratios of the radioactivities taking the values of MCF-7 to be 100 as reference. cNot detected.
of the two types of ER mRNA cannot be estimated by the RTPCR method. Among the three mammary tumor cell lines, the different transcriptional regulations of ER are suggested by the observed different levels of ER mRNAs from the distinct promoters. Therefore, it is of much interest to investigate which transcriptional regulation is responsible for the ER expression in human mammary tumors. Expression of ER mRNAs in mammary tissues of patients with breast cancer To investigate the ER expression and the use of distinct promoters, we collected samples of tumor, surrounding tissue and normal tissue from 50 patients with breast cancer, and total RNA was prepared from each of the tissue samples. Figure 3 shows the distribution of ER protein levels, assayed by enzyme immuno-assay (EIA), in tumor tissue among the 50 patients in order of ER levels. Approximately one-third of patients were ER-negative, the others being ER-positive. Of the ER-positive patients, half showed relatively low levels of ER protein, while the other half showed great variation, ranging 100–700 fmol/mg cytosol protein. Figure 4 typifies semiquantitative RT-PCR on total ER, type A, and type B mRNA, for four patients. The tumors of two patients with high ER 462
protein levels showed extremely high levels of total ER mRNA expression, together with remarkably enhanced expression of type B mRNA. However, ER mRNA expression in an ERnegative tumor was undetectable, while a tumor at a low level of ER protein also had a low level of ER mRNA expression. Next, out of the 50 patients, we selected the samples of 12 patients for the present analysis as indicated in Figure 3, i.e. eight patients with breast cancer at high ER levels, two patients at low ER levels and two patients with ER-negative breast cancer. Relative expression levels of total ER mRNA, type A and type B mRNA were then determined for tumor, plus surrounding and normal tissue from each patient. The results are summarized in Table I together with clinical characteristics of patients. Premenopausal (post-menopausal) status was associated with lower (higher) expression of total ER mRNA and typeB mRNA, but not type A mRNA, in tumors, i.e. mean relative levels 6 SE of total ER, type A and type B mRNA for premenopausal (post-menopausal) patients were 68.2 6 29.3 (169.7 6 43.9, P 5 0.08 for mean difference), 13.6 6 11.3 (15.2 6 5.7, P . 0.9, not significant), and 58.2 6 32.3 (231.3 6 55.0, P , 0.05), respectively. Relative levels of total ER mRNA in tumors were compared
mRNA expression in breast cancer
with ER protein levels (correlation coefficient 0.016, P . 0.9). On the other hand, the expression of type B mRNA was enhanced at higher levels in ER-positive tumors than that in MCF-7 cells (Figure 5C), and it showed a high correlation with ER protein levels (correlation coefficient 0.746, P , 0.005). Discussion
Fig. 5. Relative expression levels of total ER mRNA (A, left graph) type A mRNA (B, left graph), and type B mRNA (C, left graph) in tumor, surrounding and normal tissue on the 12 patients in order of ER protein levels, taking the expression levels of MCF-7 to be 100 as reference. Correlations of these mRNA expression levels in tumors to ER protein levels were examined in the right diagrams of A, B, and C.
Fig. 6. Type B mRNA expression levels in tumors and size of tumors among premenopausal (u) and post-menopausal (j) patients with breast cancer. Sizes of tumors were calculated as products of three major axes (X*Y*Z).
with ER protein levels to examine the reliability of our semiquantitative RT-PCR method, resulting in close correlation with the ER protein levels assayed by EIA in Figure 5A: a linear and positive correlation was observed between total ER mRNA levels and the protein levels (correlation coefficient 0.946, P , 0.001). Then, mRNA expression of types A and B among these patients was studied to examine which type induces enhanced ER expression in tumors, in terms of correlation analysis with ER protein levels. Type A mRNA in tumor tissues, which was expressed at much lower levels than in MCF-7 cells (Figure 5B), showed no significant correlation
ER expression plays an important role not only in the growth and differentiation of the mammary gland, but in the biology of breast cancer. Induction and/or loss of ER expression in tumors seems to be intimately associated with the progression of breast cancer. It is then important to study the transcriptional regulation of ER and clarify the mechanisms of induced ER expression seen in many breast tumors. Our results indicate that the expression of type B from the distal promoter (upstream of the proximal one) is more responsible for the over-expression of ER protein in human breast tumors than that of type A from the proximal one, although the absolute levels of these two types of ER mRNA cannot be estimated by our experiments. Induced ER expression in tumors has been reported to be associated with menstral status, suggesting that lower concentrations of estrogen around tumors in post-menopausal women result in increased expression of ER in tumors (32). The association of type B mRNA, not type A, with menstral status in Table I suggests that estrogen environment around tumors influences the transcription of ER from type B promoter, which is predominantly used in ER-positive tumors. A recent study has quantified the expression of the two transcripts in normal mammary gland, demonstrating that both types of mRNA are expressed at comparable levels (27). In addition, a very recent study using transgenic mice has shown that a tissue-specific and developmental regulation of ER is entirely owing to promoter B (33). These studies indicate the possibility that the induction of type B mRNA might occur in some developmental stages of human mammary gland in vivo by the same regulation system as that working in breast cancer. A further investigation will be required to elucidate the molecular mechanisms of regulating ER expression in relation to the progression of breast cancer. There are several candidate sequences of potential ciselements in promoter B such as E-box, AP-1, and NF-kB binding sites, which are important and responsible for cytokineand growth factors-stimuli. The induction of ER may occur as a result of change or modification in the above transcription factors in tumor cells, accompanied by promotion or progression of tumor, providing a clue to understanding the important role of estrogen in mammary carcinogenesis. Our results suggest that the specific enhancement of promoter B can be a good indicator for diagnosis and prognosis of human breast cancer. An example of this line is given in Figure 6, where increased size of tumors among post-menopausal patients is significantly associated with decreased levels of type B mRNA expression (correlation coefficient –0.864, P , 0.05), while tumor size increase among premenopausal ones is not. Further studies will be required to investigate the association with various clinical features on the basis of increased numbers of study subjects and also in combination with other molecular markers. Acknowledgements We thank Dr Masakazu Toi for providing mammary tumor cell lines and for his helpful discussion, and Ms Kyoko Hajiro-Nakanishi for her excellent
S.-I.Hayashi et al. technical assistance. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, a research grant from the Ministry of Health and Welfare of Japan.
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