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RECEPTORS |
-Receptor
Department of Biochemistry (K.-H.L., W.-J.W., Y.-H.W.), Chang-Gung University, Taoyuan, Taiwan, Republic of China; and Gene Regulation Section (S.-y.C.), Laboratory of Molecular Biology, Combined Cancer Research Center, National Cancer Institute, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: K. H. Lin, Chang-Gung University, Taoyuan, Taiwan, Republic of China. E-mail: khlin{at}mail2000.com.tw
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Abstract |
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is expressed at different levels, were used as a tool to assess the role of ER in Nm23-H1 gene-mediated metastasis. E2 induced time-dependent increases in the abundance of Nm23-H1 mRNA and protein, with the extent of these effects correlating with the level of expression of ER. E2 induced a marked decrease in the invasive activity of MCF-7 and BT-474 cells but had no effect on BCM-1 cells, which had virtually no ER. Consistent with these results, the ER-mediated Nm23-H1 promoter activity was inhibited 3-fold by the E2 antagonist, ICI 182,780. Deletion analysis of the promoter region of the Nm23-H1 gene identified a positive estrogen-responsive element located in -108/-94. ER protein bound specifically to the -108/-79 fragment with high avidity. These results indicate that E2, acting through ER, activated transcription of the Nm23-H1 gene via a positive estrogen-responsive element in the promoter region of the gene. These results suggest that E2 could suppress tumor metastasis by activating the expression of the Nm23-H1 gene. |
Introduction |
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Steroid hormones, such as E2, modulate gene expression via intracellular receptors that belong to members of the steroid hormone and retinoic acid superfamily of ligand-dependent transcription factors. E2 binds to ERs to exert its physiological responses (5, 6, 7, 8, 9). Two ER isoforms, ER and ERß, have been identified (10, 11). These two ER isoforms are derived from two separate genes and have distinct tissue expression patterns (10, 11). Accumulating evidence suggests that these two ERs have different biological properties (11). ERs bind to specific DNA sequences, termed as estrogen-responsive elements (EREs), in the regulatory regions of target genes (12, 13, 14, 15). The ERE is an inverted repeat of the sequence AGGTCA with three nucleotides separating the two half-sites. Several studies suggested that ERs bind to ERE as a homodimer in physiological conditions. This is different from TRs, which bind to DNA, both as homodimer and heterodimer, with retinoic X receptors and other members of the receptor superfamily (16, 17, 18, 19). Although much progress has been made in our understanding of the transcriptional regulation of ER target genes, little is known of the role of ERs in tumor metastasis (20, 21, 22, 23, 24).
The metastasis-associated gene Nm23-H1, which encodes an 18-kDa nucleoside diphosphate kinase (NDPK), was identified by a differential hybridization approach in K-1735 murine melanoma cells with high and low metastatic potential (25, 26). However, many studies suggest that in vitro NDPK activity of Nm23-H1 might be unrelated to its metastasis suppressor function. Two human Nm23 genes (H1 and H2) have been cloned that share 88% homology at the amino acid level (27). The Nm23-H2 gene encodes the c-MYC transcription factor PuF, which is not associated with metastasis (28). Both Nm23-H1 and Nm23-H2 have been mapped to human chromosome 17q11–21 (29). The abundance of Nm23-H1 mRNA is markedly reduced in highly metastatic tumor cell lines such as K-1735 melanoma cells (25) and Ras- and adenoviral E1A-transfected rat embryo fibroblasts cells (30), as well as in human primary breast, hepatocellular, gastric, ovarian, and cervical carcinomas (31). The expression of Nm23-H1 in malignant melanoma has been shown to be a predictive prognostic parameter for survival (32).
Our previous study indicated the T3-induced time-dependent decreases in the abundance of Nm23-H1 mRNA and protein correlate with the level of expression of TRs. T3, acting through TRs, inhibits transcription of the Nm23-H1 gene, and this effect is mediated by a negative regulatory element in the promoter region of the Nm23-H1 gene (33). In the present study, we investigated the regulation of antimetastatic Nm23-H1 gene by E2, to understand the role of estrogens in tumor metastasis. We focused our study on the ER-mediated pathway by using three cell lines that express endogenous ER at different levels but have barely detectable ERß (34). We show that the expression of Nm23-H1 was induced by E2 that is mediated, at least in part, at the transcriptional level via ER. Importantly, a functional positive ERE in the promoter region of the Nm23-H1 gene was identified. These results suggest that ER could play an important role in tumor metastasis.
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Materials and Methods |
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Western blot analysis
Western blot analysis was carried out as described (33). After transferring, the blots were incubated with rabbit polyclonal antibodies against Nm23-H1 (1:1000 dilution in TBS; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse monoclonal antibody to ER or tubulin (1:1000 dilution in TBS) for 1 h. The immune complexes were then visualized by chemiluminescence with an ECL detection kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). The intensities of immunoreactive bands were quantified by analysis with Image Gauge software (Fuji Photo Film Co., Ltd. Film, Tokyo, Japan).
Determination of the trans-activation activity of ER
E2-dependent trans-activation activity of endogenous ER was assayed in the three breast carcinoma cell lines as described (35). Briefly, cells were transfected with the reporter plasmid (2 µg) containing the ERE-luciferase, as well as with a ß-galactosidase expression plasmid (1 µg) to control for transfection efficiency. Transfected cells were subsequently incubated for 24 h in E2d medium containing various concentrations of E2, after which the activities of luciferase and ß-galactosidase in cell lysates were measured (36). The activity of luciferase was normalized on the basis of the activity of ß-galactosidase.
Northern blot analysis
Total RNA was extracted from cells with the use of TRIzol Reagent (Life Technologies, Inc.). Equal amounts of total RNA (20 µg) were analyzed on a 1.2% agarose-formaldehyde gel and subjected to Northern blot analysis as described (37). The membrane was probed with the full-length Nm23-H1 cDNA fragment (746 bp) that was amplified and labeled with [-32P]deoxy-CTP (3000 Ci/mmol; Amersham Pharmacia Biotech) by the PCR. The membrane was subsequently stripped and reprobed with a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment to verify an equal loading of RNA to each lane. To determine the cycloheximide (CHX) effect, MCF-7 and BT-474 cells were treated with CHX (10 µg/ml) for 24 h in the presence or absence of E2 (10 nM). After treatment, the RNA was isolated and analyzed as described above.
In vitro assay of invasive activity
The effect of E2 on the invasive activity of three breast carcinoma cell lines was examined with a rapid in vitro assay as described (Transwell method) (38, 39). Cell density was adjusted to 1 x 105/ml, and 200 µl of the suspension were added to each of triplicate wells. The medium in the upper chamber was serum-free DMEM supplemented or not with 10 nM E2, and that in the lower chamber was E2d supplemented with 10% FBS and with or without 10 nM E2. After incubation for 20 h at 37 C, the number of cells that had traversed the filter to the lower chamber was counted, and was then expressed as a percentage of the total number of cells to provide an index of invasive activity.
Cloning of the Nm23-H1 promoter and assay of promoter activity
Fragments of the Nm23-H1 promoter were amplified by the PCR on the basis of the published nucleotide sequence (40), which were then inserted into the pGL2 vector (Promega Corp., Madison, WI). Various deletion mutants of the Nm23-H1 promoter were constructed (see Fig. 8
) based on PCR amplification. The sequences of all promoter constructs were confirmed by automated DNA sequencing.
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1–2 x 105 per 60-mm dish) with 1.6 µg of the pGL2 vector containing Nm23-H1 promoter sequences with the use of LipofectAMINE (Life Technologies, Inc.). Cells were also transfected with 1.6 µg pcDNA3 expression vectors for ER as well as with 1.2 µg of the ß-galactosidase expression vector, pSVß plasmid (CLONTECH Laboratories, Inc., Palo Alto, CA). Twenty-four hours after transfection, the cells were incubated in the absence or presence of 10 nM E2 for 24 h and then lysed for measurement of luciferase and ß-galactosidase activities (33).
EMSA
A 32P-labeled oligonucleotide was prepared as described previously (33). For EMSA, an equal amount of ER protein was incubated with 32P-labeled ERE oligonucleotide. Protein-oligonucleotide complexes were detected by PAGE and autoradiography as described previously (33).
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Results |
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proteins in breast carcinoma cell lines proteins, we prepared cell lysates and performed Western blot analysis. As shown in Fig. 1A, the extent of expression of Nm23-H1 protein in the three cell lines was MCF-7 > BT-474 > BCM-1. The expression of ER protein was most abundant in MCF-7, less in BT-474, and almost nondetectable in BCM-1 cells. An equal amount of tubulin was detected in these three cell types, indicating an equal loading of proteins for Western analysis of Nm23-H1 and ER proteins. These results indicate that the expression of Nm23-H1 and ER proteins is cell-type-dependent and is positively correlated between these two proteins.
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in MCF-7, BT-474, and BCM-1 cells. MCF-7 and BT-474 cells exhibited high trans-activation activity, which was increased by E2 in a concentration-dependent manner (Fig. 1B
). BCM-1 exhibited virtually no detectable activity, consistent with the lack of detectable ER (Fig. 1A). These results indicate that the level of expression of ER protein correlates with the extent of trans-activation activity and that the ER is functional in MCF-7 and BT-474 cells.
Effects of E2 on the abundance of Nm23-H1 mRNA and protein in breast cancer cell lines
The effect of E2 on the expression of the Nm23-H1 protein (18 kDa) was compared among the three breast carcinoma cell lines by treating the cells with E2 for increasing lengths of time (Fig. 2). Western blot analysis reveals that the exposure of MCF-7 and BT-474 cells to 10 nM E2 resulted in a time-dependent increase in the amount of Nm23-H1 protein (Fig. 2A). Compared with untreated cells, E2 increased the abundance of Nm23-H1 in MCF-7 and BT-474 cells at each time point but not in BCM-1 cells. The intensities of these bands were quantified and normalized against the intensities of tubulin. The normalized data are shown in Fig. 2D. The amount of Nm23-H1 was increased by 62, 85, and 106% after incubation of MCF-7 cells with 10 nM E2 for 6, 12, and 24 h, respectively (Fig. 2D), whereas it was increased by 27, 138, and 124% in BT-474 cells after treating with E2 for 12, 24, and 48 h, respectively (Fig. 2D). In contrast, the expression of Nm23-H1 protein in BCM-1 cells was largely unaffected by E2 (Fig. 2D).
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, A–C). The intensities of mRNA bands were quantified and normalized against the intensities of GAPDH (Fig. 3D). Exposure of cells to 10 nM E2 resulted in a time-dependent increase in the amount of Nm23-H1 mRNA (Fig. 3D), with increases of 85, 110, and 98% in MCF-7 cells and 235, 345, and 310% in BT-474 cells after incubation of cells with E2 for 12, 24, and 48 h, respectively (Fig. 3D). No apparent effect of E2 on the expression of Nm23-H1 mRNA in BCM-1 cells was detected (Fig. 3D). Thus, at least part of the effect of E2 on the expression of Nm23-H1 mRNA seems to be mediated at the transcriptional and/or mRNA level, leading to the increased expression of Nm23-H1 protein.
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, inhibition of de novo protein synthesis, by treating the cells with CHX, had no effect on the basal (lanes 1 vs. 2 of Fig. 4) or on the E2-induced activation (lanes 3 vs. 4 of Fig. 4) of Nm23-H1 mRNA in MCF-7. Similar results were also obtained for BT-474 cells (compare lanes 5 vs. 6 and lanes 7 vs. 8). These data indicate that the increase in the Nm23-H1 mRNA was at the transcriptional level as a result of direct action of E2.
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). Incubation with E2 reduced the invasive activity in MCF-7 cells approximately 2.5-fold. E2 reduced the invasive activity of BT-474 cells approximately 2-fold, but it had no effect on that of BCM-1 cells. The marked E2-induced decrease in the invasive activity of MCF-7 and BT-474 cells was thus consistent with the high level of ER expression and the largely-activated effect of E2 on the expression of Nm23-H1 protein in these cells.
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, at 10 nM E2, Nm23-H1 promoter had the highest activity (approximately 4-fold activation). However, at a 10-fold-higher E2 concentration (100 nM), a 25% reduction was detected (Fig. 6). Therefore, the subsequent determinations of the E2-dependent trans-activational activity were carried out using 10 nM E2.
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signal transduction pathway on Nm23-H1 promoter is E2-dependent, we used an E2 antagonist, ICI 182,780, to determine whether it blocked the trans-activation activity of ER mediated by Nm23-H1 promoter (Fig. 7). As shown in Fig. 7, the antagonist, ICI 182,780, inhibited 82% of the E2-dependent ERE-mediated trans- activation activity. Figure 6 further shows that a similar extent of inhibition (77%) by ICI 182,780 on the E2-dependent Nm23-H1 promoter-mediated trans-activation activity was detected. These results provide additional support that the expression of Nm23-H1 gene is transcriptionally regulated by E2.
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interaction site on the Nm23-H1 promoter, we carried out deletion analysis. Vectors containing a series of truncated Nm23-H1 promoter sequences were constructed and transfected into MCF-7 cells. Fig. 8 shows the trans-activational activity of the -528/+69 (+1 is relative to the transcriptional initiation site) containing the reporter construct (Reporter I, Fig. 8) was increased 3.5-fold in the presence of E2. When the 3' end of the -528/+69 was truncated to yield the -528/-228 (Reporter II) and -258/-68 (Reporter III) constructs, the E2-induced activation was substantially reduced (by 91% and 85% of activity, respectively). Cells transfected with -108/+69 construct (Reporter IV) led to a 3.5-fold transcriptional activity, compared with -258/-68 construct, suggesting the existence of repressor activity in -258/-109. These data further indicate that the ER interaction site was localized in the -108/+69 region. The localization of the ER interaction site in this region was further confirmed by using a different reporter construct that contained a TK promoter upstream of Luc reporter (Reporter IV vs. V, Fig. 8). To further localize the promoter sequence responsible for the E2-induced trans-activation activity, we divided -108/+69 into four fragments, with the sequences encompassing -108/-32, -68/+32, -8/+69, and -108/-68 (reporters VI –IX) and compared their trans-activation activities. Compared with -108/+69, deletion of some of the 5' upstream sequences of -68 led to a significant drop of activity, as shown in the reduced activities exhibited by promoters containing -68/+32 (reporter VII) and -8/+69 (reporter VIII) regions. There were no significant differences in the activities exhibited by -108/-32 (Reporter VI) and -108/-68, which has a shorter sequence (Reporter IX). We therefore concluded that the positive ERE was located in the -108/-68 region, which does not contain the AP1 site.
Examination of the nucleotide sequences in the -108/-68 region of the Nm23-H1 promoter reveals an ERE with the sequence of -108(TAACCG)gaa(AGGTCT)-94 (Fig. 9B). It is homologous to the ERE consensus sequence (AGGTCA)gag(TGACCT) reported by Driscoll et al. (13), except with the exchange of left- and right-half sites.
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-containing lysates, respectively; Fig. 9). The binding of ER to -108/-79 fragment was reduced in the presence of 10-fold molar excess of unlabeled specific competitor (lane 4; SC) but not in the presence of 10-fold molar excess of nonspecific competitor (lane 5; NSC, with irrelevant sequences). These results indicate that the DNA-bound ER bands shown in lanes 2 and 3 were specific.
The core sequence in the -108/-94 in the Nm23-H1 promoter was TAACCG gaaAGGTCT (Fig. 8B). We mutated the wild-type sequence to TAACCGgaaCGGTCC (Mutant 1) and CACCCGgaaAGGTCT (Mutant 2) to further confirm the specificity of the ER binding site on Nm23-H1 promoter (Fig. 9, A and B). Increasing amounts of in vitro-translated ER were used in EMSA (2 and 4 µl ER-containing lysates). The intensities of the ER bound-ERE complex were quantified. As shown in Fig. 9C, Mutant 1 and Mutant 2 lost 72% and 75% of the binding activity of the wild-type ERE, respectively, in the presence of either 2 or 4 µl of the in vitro-translated ER. These results indicate that both half-sites are important for the binding of Nm23-H1 promoter to ER. Taken together, these findings indicate that the two ERE half-site binding motifs, when exchanged from left to right sides, are functional.
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Discussion |
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, allowed us to study the possible regulatory role by E2 of the Nm23-H1 gene in tumor cell invasion. The differential expression of ER in these cell lines reveals that the extent of the up-regulation of the Nm23-H1 gene by E2 depends on the expression level of ER. The regulation of the Nm23-H1 gene by E2 is mediated, at least in part, at the transcriptional level, clearly as a result of a direct interaction of ER with the promoter region of the Nm23-H1 gene.
One functional consequence of the induction of the Nm23-H1 gene expression by E2 was an inhibitory effect on the invasive activity of MCF-7 and BT-474 cells. Those two cell lines express functional ER at different levels. The extent of the E2-induced decrease in invasive activity correlated with the extent of ER expression in these cells. The inhibitory effect of E2 on cancer cell invasion and motility has also been reported by others (20, 41, 42). These studies showed that E2 inhibits the invasion and motility in breast and ovarian cancer cells expressing ER. Furthermore, ER-negative cells (e.g. MDA-MB231, 3Y1-Ad12), when stably transfected with ER, have lower motility and invasion in the presence of E2 (20, 41, 42). It is also known, however, that estrogens promote the proliferation of ER-positive cells both in culture and in nude mice (43, 44). Thus, estrogens in ER-positive breast cancer cells have a dual effect, because they stimulate tumor growth but inhibit invasion and motility. Our present results not only are consistent with the findings by others (20, 41, 42) but also provide a viable mechanism to account for the decreases in motility and invasion in ER-positive cancer cells.
Even though the extent of the invasive activity determined in vitro may not reflect completely the in vivo metastasis, the critical role of Nm23-H1 in the motility and invasion of cancer cells has been supported by the functional studies on several Nm23-H1 mutants (45). MacDonald et al. (45) found that mutations of proline 96 and serine 120 of Nm23-H1 abrogate the motility inhibitory activity of the transfected cells, thus directly providing a biochemical basis for its metastatic inhibitory phenotype. The correlation of reduced Nm23-H1 expression and increased invasion observed in model cell systems is also confirmed by studies in patients with breast cancers. In primary tumors, a negative trend between degree of local invasion and level of Nm23-H1 expression was detected (46). A further decrease of nm23-H1 expression was detected in the invasive tumors that metastasize to axillary lymph nodes (46). An examination of 168 breast carcinomas shows that Nm23-H1 expression is correlated with longer metastasis-free survival in both node- positive and node-negative patients (47). Recently, in a cohort study of 168 breast cancer patients, a high Nm23 expression was found to be associated with the absence of distal metastases (48).
The sequence requirements in ERE for binding to ER were evaluated by Driscoll et al. (13). They reported that the minimal consensus sequence for ERE is GGTCAgagTGACC. A single base change in the consensus sequence may lead to the loss of binding to ER. For example, the ER binding affinity drops dramatically if the half-site GGTCA is changed to GGGCA. However, this change can be rescued by adding A to the 5' end of the consensus sequence (AGGGCA). Therefore, changes in the nonconsensus flanking sequences of ERE can greatly alter the extent of the interaction of ER with ERE (13). The arrangement of half-site in the 5' flanking region of the Nm23-H1 promoter is in the reverse order of that in the ideal ERE. However, it is also an inverted repeat with three spacing between the half-site binding motifs. The ER binding affinity decreased dramatically by changing 2 nucleotides in one of two half-sites from AGGTCT to CGGTCC (-99/-94) or TAACCG to CACCCG (-108/-103). These results indicate that both half-sites are critically important for ER binding. However, our in vitro DNA binding and trans-activation studies clearly indicate that an inverted motif in Nm23-H1 promoter is capable of mediating the specific activation of ER by E2.
At present, however, it is not clear whether ERß could also interact with the positive ERE identified in the promoter of Nm23-H1 gene, to affect its expression. The endogenous ER present in MCF-7 and BT-474 is mainly -subtype, with relatively very low ERß (34). The complete lack of E2- dependent trans-activation activity in BCM-1 cells indicates the absence of both ER and ERß in these cells. However, ER and ERß have DNA binding domains that are virtually identical except for one amino acid residue (10, 11). Therefore, it is reasonable to expect that ERß would bind to the positive ERE in the promoter of Nm23-H1 gene. However, because sequences of the A/B domains and the activation function-1 regions of these two ER isoforms are quite different (11), in spite of their similar high affinity to E2 (49), their transcriptional activation of Nm23-H1 promoter could be different. This could lead, to different extents, to the modulation of E2-mediated decreases in the invasive activity of cancer cells. The role of ERß in the regulation of Nm23-H1 gene and metastasis of cancer cells will await future studies.
Despite its importance in tumor metastasis, little is known about the regulation of expression of the Nm23-H1 gene. Linoleic acid and arachidonic acid inhibit the expression of Nm23-H1, whereas 
-linolenic acid increases the expression of the protein (50). The expression of the Nm23-H1 gene has also been shown to be reduced by vitamin D (51), TNF-, and interferon- (52). However, the underlying molecular mechanisms for these effects are unknown. The 5' region of the human Nm23-H1 gene shows the presence of motifs typical for transcriptional elements such as TFIID, AP-1, and CTF/NF1. A common transcription initiation site is located at -136 upstream from the first ATG codon in several tumor cell lines (40). Chen et al. (40) showed that the presence of AP-1 and CTF/NF1 elements are essential for promoter activity. We have previously reported that T3, acting through TRs, inhibits transcription of Nm23-H1 gene and that this effect is mediated by a negative regulatory element in the promoter region of the gene (33). Based on these studies, we postulate that the Nm23-H1 in these three breast carcinoma cells could also be regulated by T3. However, the expression of TR in these three breast carcinoma cells is undetectable (data not shown). Thus, the regulation of Nm23-H1 gene expression depends on the types of receptors and hormones present in the microenvironment. Our results show that E2, via its -receptor, positively regulates the expression of the Nm23-H1 gene at the transcriptional level. Thus, the present study identifies ER, another ligand-dependent transcription factor that directly interacts with a gene that plays an important role in tumor metastasis.
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Footnotes |
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Abbreviations: CHX, Cycloheximide; E2d, E2-depleted; ERE, estrogen responsive element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received August 2, 2001.
Accepted for publication October 10, 2001.
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2: role of the ninth heptad in DNA binding, heterodimerization with retinoid X receptors, and dominant negative activity. J Biol Chem 271:28235–282421 and ß1 receptors in a human hepatocellular carcinoma cell line. Endocrinology 137:4073–4081[Abstract]
1 receptors from patients with hepatocellular carcinoma. Endocrinology 138:5308–5315 and ß. Endocrinology 138:863–870This article has been cited by other articles:
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