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Molecular Biology, Pathobiology, and Genetics |
1 Medical Molecular Biology Unit, Institute of Child Health, University College London; 2 Cancer Research UK, Hedley Atkins Breast Pathology Laboratory, Hedley Atkins Breast Unit, King's College London, Guy's Hospital; and 3 Department of Cardiology, Cardiovascular Division, GKT School of Medicine, The Rayne Institute, St. Thomas's Hospital, London, United Kingdom; and 4 Department of Genetics, Harvard Medical School, Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts
Requests for reprints: Vishwanie S. Budhram-Mahadeo, Medical Molecular Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, United Kingdom. Phone: 44-207-2429782; Fax: 44-207-9052301; E-mail: v.mahadeo{at}ich.ucl.ac.uk.
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Introduction |
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Changes in genes that are expressed in breast cells can significantly affect the proliferation and behavior of these cells and contribute to development and progression of neoplasms. For instance, increasing levels of genes, such as c-myc (1), cyclin D1 (2), and estrogen receptor (ER; ref. 3), are associated with increased proliferation in many breast cancers, whereas high expression of genes, such as HSP-27 and cathepsin D, are associated with increased motility, invasion, metastasis, and/or drug resistance (4–9). Transcription factors that control the expression of these genes in a tissue-specific manner or in response to specific signals can profoundly affect the behavior of these cells.
We have been investigating the role of the Brn-3b POU transcription factor in breast cancers. This protein is elevated in many breast cancers compared with levels in normal breast epithelial cells and seems to play an important role in determining proliferation and growth characteristics of breast epithelial cells (10, 11). Brn-3b belongs to the class IV POU domain family (12, 13) and is expressed in developing and adult nervous system (14, 15) as well as in adult reproductive tract tissues (breast epithelial cells, ovary, and testis; ref. 11). Brn-3b mRNA and protein are expressed at high levels in biopsies taken from patients with breast cancers compared with those from benign breast diseases (11). Two isoforms of Brn-3b exist as a result of alternative promoter usage (13). The longer 43-kDa Brn-3b(l) protein contains an additional domain at the amino terminus, which is absent in the shorter 30- to 35-kDa Brn-3b(s) isoform. Both the long and the short forms of Brn-3b have been detected in breast cancer cells (11).
Studies undertaken using stable cell lines generated to either overexpress Brn-3b(s) or decrease endogenous Brn-3b using an antisense strategy showed that changing the levels of Brn-3b can significantly modify the proliferation and behavior of cancer cells, including the human MCF-7 breast cancer cell line and neuroblastoma cell line, IMR32 (10, 16). In these cells, high levels of Brn-3b resulted in increased anchorage-dependent growth and proliferation as well as anchorage-independent colony formation in soft agar compared with a control transfected cell line (10, 16). In contrast, loss of Brn-3b using an antisense approach resulted in much slower growth in a monolayer as well as failure to form colonies in soft agar. We showed that elevated Brn-3b enhanced tumor growth in vivo in xenograft models, whereas decreased Brn-3b levels resulted in slower growing tumors in these xenograft models (16). We also showed that high levels of Brn-3b confer resistance to antiproliferative agents, such as retinoic acid, and increases the invasiveness of the neuroblastoma cells (16). These results provided direct evidence that Brn-3b, on its own, was capable of changing the growth characteristics and behavior of cancer cells.
As a transcription factor, Brn-3b will mediate its effect in mammary epithelial cells by regulation of gene expression, either directly or indirectly. For instance, Brn-3b could directly and strongly repress the activity of the BRCA1 promoter and this was paralleled by decreased levels of BRCA1 in tumors with elevated Brn-3b (11). Moreover, Brn-3b can physically interact directly with the ER and enhance its transcriptional effect on an ER element (ERE)–containing promoter (17). The ability of Brn-3b to interact with and modify the effects of proteins, such as ER, which is clearly important in determining the growth and behavior of breast epithelial cells, supports an important role for this transcription factor in breast cancer development and/or progression.
To understand how Brn-3b could mediate diverse effects in tumor growth and proliferation as well as invasiveness of cancer cells, we investigated target genes that are regulated by Brn-3b and that may contribute to these different effects by using the manipulated MCF-7 cells expressing either high or low levels of Brn-3b. We previously described cyclin-dependent kinase, CDK4 (18), as a target that was transactivated by Brn-3b in these cells. In contrast, another target gene, plakoglobin, which is associated with cell adhesion and is down-regulated in many cancers (19), is repressed by high levels of Brn-3b.5
One target gene that is of interest in breast cancer is the small heat shock protein, HSP-27, which is expressed in different cell types and is associated with diverse cellular functions, including thermotolerance, signal transduction (14), differentiation (15), and protection against apoptosis (11). Increasing evidence arising from studies using breast tumor biopsies and breast cancer cell lines now supports a role for HSP-27 in tumor invasion and resistance to chemotherapeutic drugs. Studies carried out using breast cancer cell lines overexpressing HSP-27 show increased resistance to the chemotherapeutic drug, doxycycline (4, 6). Moreover, elevated HSP-27 also resulted in increased anchorage-dependent proliferation and anchorage-independent growth, suggesting that HSP-27 can modify growth and behavior of breast cancer cells (5, 6). Analysis of HSP-27 expression in human breast biopsies supports a correlation with invasiveness and drug resistance (20, 21). Thus, increased expression of HSP-27 in breast cancers is likely to affect the prognosis and outcome of treatment.
Here, we report that changing Brn-3b levels in breast cancer cells can change the levels of endogenous HSP-27 in these cells and this is supported by a positive correlation of HSP-27 protein expression with Brn-3b in biopsies taken from patients with breast cancers. Furthermore, we showed that Brn-3b can strongly transactivate the HSP-27 promoter either alone or on association with the ER and is directly associated with the promoter in vivo. These results and the implications of this regulation of HSP-27 expression by Brn-3b are discussed.
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Materials and Methods |
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antibodies (Santa Cruz Biotechnology) both used at 1:2,000; and goat polyclonal anti-actin (I-19, Santa Cruz Biotechnology) used at 1:1,500. Horseradish peroxidase–conjugated secondary antibodies were obtained from DAKO (Cambridgeshire, United Kingdom) and used at 1:2,000 to 1:3,000. Cell lines, plasmids, and tissues. MCF-7 breast cancer cell line was obtained from American Type Culture Collection (Manassas, VA) and grown in full growth medium or stripped serum medium as described previously (17). Stably transfected MCF-7 cells expressing different levels of Brn-3b were described previously (10).
Brn-3b(l) and Brn-3b(s) cDNAs were cloned into the pLTR expression vectors, whereas the ER expression vector was a kind gift from Dr. M. Parker (Imperial College, Hammersmith Campus, London, United Kingdom; ref. 17). The short hairpin RNA interference (shRNAi) construct (si9) targeted specifically to Brn-3b was prepared using the pSHAG vector (ref. 22; a kind gift from Dr. G. Hannon, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The sequence 5'-AAAAAAGGTGGTGATAGTGGTAGTGGTAGTGGTAACAAGCTTCTCACCACCACCACCACCACCATCACCACCGGTGTTTCGTCCTTTCCACAA-3' was one of three sequences designed to target Brn-3b using the dedicated program available on the Cold Spring Harbor Laboratory Web site.
The HSP-27 reporter construct contained 225 bp of the proximal HSP-27 promoter cloned into pGL2-Basic luciferase reporter vector (Promega, Southampton, United Kingdom; ref. 23). Site-directed mutagenesis of the Brn-3 site in the HSP-27 promoter was carried out using QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using primers 5'-CGGGTCATTGCCGGCAATAGAGACCTC-3' and 5'-GAGGTCTCTCTTGCCGGCAATGACCCG-3'. This introduced a NaeI restriction site that was used for diagnostic purposes.
Breast biopsies were obtained as anonymous samples from the Hedley Atkins Breast Tissue Bank and are covered by local and national ethical approval. Invasive carcinoma was graded according to the modified Bloom and Richardson system (24).
Western blot analysis. Total cellular proteins were prepared for Western blot analysis from either cell lines (harvested directly in 2x Laemmli buffer) or breast biopsies (homogenized in liquid nitrogen and then transferred into Laemmli buffer) as described previously (11). Proteins were resolved by SDS-PAGE on 12.5% polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes (Hybond-C, Amersham, Buckinghamshire, United Kingdom). To detect specific proteins, membranes were blocked in 4% nonfat powdered milk (Marvel) in PBS (pH 7.4) plus 0.1% Tween 20 (PBST) for 1 hour and then incubated with the appropriate primary antibody at the specified dilution in 1% milk plus PBST (PBSTM) for 1 to 3 hours. Membranes were washed for 5 x 5 minutes in PBSTM without antibody and then incubated with secondary antibody in PBSTM for 45 minutes to 1 hour. Membranes were then washed for 4 x 5 minutes in PBSTM followed by 1x 5 minutes in PBST and blots were developed using enhanced chemiluminescence reagent (Amersham). Blots were scanned and bands were quantified using Bio-Rad (Hertfordshire, United Kingdom) GS-800 scanning densitometer. Differences in total protein levels were adjusted by using the appropriate actin levels and these are represented in the semiquantitative values shown.
Cell culture and transient transfections. MCF-7 breast cancer cells were maintained in DMEM supplemented with 10% fetal calf serum plus 1% nonessential amino acids and 1% penicillin/streptomycin. For transient transfection experiments, cells were grown under special conditions to reduce endogenous ER levels [i.e., in DMEM without phenol red and dextran-coated charcoal-stripped fetal calf serum (17)]. Cells were plated into six-well plates at a density of 5 x 104 cells per well and transferred into stripped serum medium for 72 hours before transfection experiments. Transfections were carried out with the appropriate reporter/expression vectors specified using Fugene (Roche, Herts, United Kingdom) according to manufacturer's protocol.
Luciferase reporter assays. Forty-eight hours following transfection, cells were harvested in 250 µL of 1x passive lysis buffer (dual-luciferase reporter assay kit, Promega) after washing with PBS. Cells were lysed for 15 minutes at room temperature, and cell lysates were transferred to 1.5 mL microcentrifuge tubes, vortexed briefly, and centrifuged at 13,000 rpm for 5 minutes to pellet cell debris. Supernatants were transferred to clean tubes and used for analysis of promoter activity using dual-luciferase reporter assay kit and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). Measurement of firefly luciferase activity was followed by measurement of control Renilla luciferase using the "Stop and Glo" reagent and this was used to control for variations in transfection efficiency. This was carried out according to the manufacturer's protocol. Values were expressed as a percentage of the empty long terminal repeat (LTR) vector control.
Electrophoretic mobility shift assay. dsDNA sequence (100 ng) corresponding to the Brn-3 binding site in the HSP-27 promoter (5'-CGGGTCATTGCCATTAATAGAGACCTC-3'; ref. 25) was labeled with [
-32P]ATP using T4 kinase. To prepare the labeled probe for use in electrophoretic mobility shift assay (EMSA), unincorporated [-32P]ATP label was removed using a Sephadex G-25 column. The probe (2 ng) was incubated in all reactions, which included cellular extracts prepared from the breast cancer cell line MCF-7 overexpressing Brn-3b. To test for specificity and affinity of Brn-3b protein binding to the labeled probe, different competitors were used. Specific competition was carried out using 100x cold unlabeled Brn-3 site. The oligonucleotide corresponding to the ERE was used as nonspecific competitor (26). The oligonucleotide containing the mutated Brn-3 site (5'-CGGGTCATTGCCGGCAATAGAGACCTC-3') that was introduced into the mutated promoter was also used to test whether it could compete for binding to Brn-3b. Brn-3b polyclonal antibody (2 µL, Santa Cruz: Insight Biotechnology) was used to supershift the protein complex.
Chromatin immunoprecipitation assay. This technique was carried out as described by Gascoyne et al. (27). Briefly, Brn-3b-overexpressing MCF-7 cells or control cells were used for these studies. Cross-linking was done by addition of 270 µL of 37% formaldehyde to 10 mL of the medium and incubation for 15 minutes. Cells were washed, harvested in cold PBS, and then lysed in lysis buffer [1% SDS, 0.01 mol/L EDTA, 0.05 mol/L Tris (pH 8.0), and 0.01% protease inhibitor cocktail]. The cells were sonicated to shear the DNA and then subjected to centrifugation. A small volume of the supernatant was taken out for use as input sample in subsequent PCR analysis, whereas the remaining sample was divided and then subjected to immunoprecipitation overnight using the appropriate antibody [either Brn-3b goat polyclonal antibody (Santa Cruz Biotechnology) or secondary anti-goat antibody (DAKO)]. Incubation with protein G-Sepharose beads allowed immobilization of protein-antibody complex that was washed thoroughly and then eluted from the beads. The cross-links between the protein and the DNA were reversed by incubation at 65°C for 4 hours and the proteins were digested using proteinase K. Following phenol/chloroform extraction, the DNA was precipitated with ethanol, resuspended in water, and used for PCR. The primers used in the PCR to amplify the promoter containing the Brn-3 site are HSP-27F1 5'-AACGAGAGAAGGTTCCAGATG-3' and HSP-27R 5'-CTCCAGTCGGGTATTTTTAGC-3'. Standard conditions were used for PCR amplification and included the use of 2.5 mmol/L MgCl2 with the following cycling variables: 1 cycle at 94°C for 15 minutes followed by 40 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds. The reaction was completed by an incubation of 72°C for 5 minutes. PCR products were resolved on a 2.5% agarose/Tris-borate EDTA gel.
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Results |
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Figure 1A shows that elevated HSP-27 levels were observed in MCF-7 cells (+) that overexpress Brn-3b compared with vector control transfected cells, whereas HSP-27 levels were decreased in antisense cells with reduced Brn-3b. We measured the changes in HSP-27 protein levels by scanning densitometry and used actin to equalize for variation in protein loading. As shown in Fig. 1B, a 1.3- to 1.4-fold increase was observed in Brn-3b-overexpressing cells compared with the vector controls (paired t test; P = 0.05), whereas a reproducible decrease of almost 50% of the endogenous levels was seen in the Brn-3b antisense cells (paired t test; P = 0.03). This change in HSP-27 levels was directly and specifically associated with changes in Brn-3b protein expression, as HSP-70 and HSP-90 levels did not change in response to different levels of Brn-3b (Fig. 1A). Whereas this approach provides a semiquantitative measure of protein levels, the reproducible changes observed for HSP-27, but not HSP-70 or HSP-90, suggest a direct relationship of HSP-27 levels with Brn-3b protein in the breast cancer line, MCF-7.
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As shown in Fig. 3A, Brn-3b could strongly transactivate the HSP-27 promoter with increasing amounts of both Brn-3b(l) and Brn-3b(s) isoforms resulting in a significant increase in the promoter activity compared with the empty expression vector. Thus, up to 10-fold activation was observed when a 2:1 ratio of Brn-3b to promoter was transfected [paired t test, P = 0.001 and 0.0004 for Brn-3b(s) and Brn-3b(l), respectively]. Thus, high levels of Brn-3b can indeed significantly increase the activity of the HSP-27 promoter and hence its expression in breast cancer cells.
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Brn-3b is important for the estrogen receptor–mediated activation of the HSP-27 promoter. To show that the effect on the HSP-27 promoter was dependent on Brn-3b, we used RNAi in which shRNA (22) was used to abolish Brn-3b expression in the absence and presence of ER (see Materials and Methods). The results in Fig. 4 showed that cotransfection of Brn-3b expression vector with the shRNA (si9) construct (which specifically targeted Brn-3b) resulted in a decrease in the activity of HSP-27 promoter to a level comparable with that seen in control LTR-transfected cells (P = 0.0009). Another shRNA construct (si7), which targeted the related Brn-3a, failed to repress Brn-3b-mediated effects on this promoter, suggesting that these effects were specific to the si9 construct. The si9 shRNA construct, but not si7, specifically decreased Brn-3b protein expression in transfected cells (Fig. 4B). Furthermore, the internal control, Renilla luciferase reporter, was unaffected in these experiments.
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The Brn-3 site found between two 1/2 estrogen receptor elements is required for the effect of Brn-3b on the proximal HSP-27 promoter. Previous analysis of the HSP-27 proximal promoter revealed an AT-rich sequence at (–75 bp) from the transcription start site that was shown to contain a Brn-3 consensus site (23) that could bind both Brn-3a and Brn-3b proteins. This Brn-3a site, which is flanked by two 1/2 ERE sites [Fig. 5A(i)], was required for the related Brn-3a protein to stimulate the promoter activity in a neuronal cell line (23). We therefore tested whether mutation of this Brn-3 site could alter the ability of Brn-3b to transactivate the HSP-27 promoter in breast cancer cells and the impact of this on ER-mediated transcription. Cotransfection studies were therefore carried out as before but using the reporter construct in which the Brn-3 site was mutated in the HSP-27 promoter [Fig. 5A(ii)].
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9-fold increased activity on this mutant promoter compared with up to 20-fold increased promoter activity observed in the presence of the wild-type promoter under similar conditions (Fig. 3B). This suggests that binding of Brn-3b to the Brn-3 site in this promoter is required for the maximal stimulation of HSP-27 promoter in the presence of ER also. Moreover, cotransfection of Brn-3b with ER on the mutated promoter not only blocked the additive effect normally induced by coexpression of Brn-3b and ER but also resulted in a loss of the ER-mediated transactivation. Thus, the inability of endogenous or exogenous Brn-3b to bind to this promoter seems to compromise ER-mediated transcriptional activity of this promoter perhaps by interacting with and removing the ER from the promoter or by facilitating the binding of the repressor HET/SAF-A factor to the same site, which can alter ER-mediated transcription on the HSP-27 promoter (30). Brn-3b protein fails to interact with the mutated Brn-3 site. We next tested whether the loss of activation by Brn-3b on the mutated HSP-27 promoter was a consequence of lack of binding of this protein to the mutated Brn-3 site. EMSA was done using cellular extracts obtained from MCF-7 cells overexpressing Brn-3b proteins. As shown in Fig. 6A, incubation of Brn-3b-overexpressing MCF-7 cell extract with 32P-labeled oligonucleotide probe corresponding to the wild-type Brn-3 binding site resulted in formation of specific complexes (lane 1) indicated by the arrows that was competed by addition of unlabeled Brn-3 binding site (lane 2) but not a nonspecific oligonucleotide (lane 3). Furthermore, addition of an excess of the unlabeled mutant oligonucleotide (in which the Brn-3b site was mutated) failed to compete for binding of Brn-3b to the wild-type probe (lane 4). This supports the evidence that Brn-3b does not bind to the mutated DNA. Addition of Brn-3b polyclonal antibody resulted in a further retardation (supershift) of the complex (shown by asterisks; lane 5), indicating that Brn-3b was specifically bound to this site. This supershift was not observed on addition of similar amounts of the unrelated actin antibody, confirming the specificity of the interaction of Brn-3b to this site in the HSP-27 promoter. The presence of two complexes that could be supershifted on addition of the antibody would suggest that the Brn-3b protein bound to this site on its own but may also be associated with other cellular proteins, thus giving rise to the larger complex.
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Chromatin immunoprecipitation assay showing that Brn-3b associates with the HSP-27 promoter in vivo. Because we have shown that Brn-3b could modify the HSP-27 promoter by its association with a specific site in the promoter, we next used chromatin immunoprecipitation (ChIP) assay to test whether Brn-3b is associated with the HSP-27 promoter in vivo in tumor cells. For these studies, we used MCF-7 cells that stably overexpressed Brn-3b and were shown to have elevated HSP-27 protein as well as control MCF-7 cells. Primers were designed to amplify a 157-bp fragment of the promoter that encompassed the Brn-3b binding site. As can be seen from Fig. 6B, this site can be amplified using cell extract from which Brn-3b protein has been immunoprecipitated. Lane 2 indicates the input sample, whereas lanes 3 and 4 show amplification following immunoprecipitation of control MCF-7 cells or MCF-7 Brn-3b cells (which constitutively overexpressed Brn-3b) with a Brn-3b antibody. As expected, low levels of the promoter sequence were amplified from extracts prepared using control (MCF-7) cells containing endogenous Brn-3b (lane 3) but were found at much higher levels in cells that overexpressed Brn-3b (lane 4). Lane 5 shows the background levels using anti-goat antibody control in the MCF-7 Brn-3b cells. Lane 1 indicates the 100-bp DNA ladder.
These results show that Brn-3b is indeed associated with the HSP-27 promoter in vivo in MCF-7 human breast cancer cell line and as such is likely to be a key regulator of expression of this gene in these cells.
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Discussion |
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In this study, we have shown that modifying the expression of the Brn-3b transcription factor in the human breast cancer cell line MCF-7 resulted in changes in the levels of HSP-27 protein. Thus, overexpression of Brn-3b in stably transfected cells resulted in a significant increase in HSP-27 levels compared with the controls. Conversely, decreasing Brn-3b levels in these cells resulted in a decrease in HSP-27 protein levels. Analysis of 40 biopsies taken either from patients with breast cancer or from benign breast growths shows a striking correlation of Brn-3b with HSP-27 proteins. Taken together, the correlation of Brn-3b with HSP-27 protein in breast biopsies and in the manipulated cell lines suggests that expression of HSP-27 is regulated either directly by Brn-3b and/or indirectly on interaction of Brn-3b with ER.
Using cotransfection assays, we showed that Brn-3b proteins can strongly transactivate the HSP-27 promoter activity (10- to 12-fold increase). Because HSP-27 is a known target of ER, we controlled for the ER-mediated effects by carrying out all studies using cells grown in conditions known to reduce endogenous ER levels (phenol red–less medium containing stripped serum). Thus, the effects seen are attributable to a direct effect of Brn-3b on the promoter activity rather than via its interaction with ER. Moreover, we showed that coexpression of ER with Brn-3b resulted in an additive effect on transactivation of this promoter with a 35- to 40-fold induction. This effect is as expected because we have shown previously that Brn-3b interacts with the ER and enhances the transcription of an ERE-containing promoter (17).
Using RNAi to specifically target Brn-3b mRNA, we successfully blocked the transactivation observed on transfection with Brn-3b. This confirms that Brn-3b can indeed directly transactivate the HSP-27 promoter. More interestingly, we found that targeting Brn-3b expression using RNAi resulted in a decrease in the transcriptional activity by the ER alone or when coexpressed with Brn-3b. Such results would suggest that endogenous or exogenous Brn-3b can significantly influence ER-mediated transcription on the HSP-27 promoter.
To investigate the mechanism underlying this effect, we analyzed the regulatory region of the HSP-27 gene used in this study. This consisted of a 200-bp proximal promoter sequence that contained several important and conserved elements required for efficient regulation of transcription of the HSP-27 gene (29). We have shown previously that an AT-rich sequence found at –75 bp relative to transcriptional start site contained the Brn-3 consensus site. Using site-directed mutagenesis of bases within this site, we could abolish the activation of this promoter by the related Brn-3a transcription factor in neuronal cells (23) but did not affect basal promoter activity (as the TATA box was assigned to a region –25 bp relative to start site). In this study, we showed that the HSP-27 promoter containing the mutated site failed to bind Brn-3b protein and is not transactivated by the Brn-3b proteins. Whereas transfection of ER alone continued to transactivate the promoter, this effect was attenuated compared with the ER-mediated activity observed on the wild-type promoter and addition of Brn-3b did not produce any additional activation. The ability of Brn-3b to bind to the putative site was confirmed in EMSAs in which Brn-3b could bind to the wild-type DNA sequence but not the mutated sequence. Furthermore, we have used ChIP assay to show that Brn-3b does bind to this region of the HSP-27 promoter in vivo as well as in vitro.
These findings would suggest that binding of Brn-3b to the consensus site is sufficient to transactivate the HSP-27 promoter. However, it also seems that Brn-3b is necessary for maximal stimulation of this promoter by ER, as coexpression of Brn-3b with ER on the mutated promoter abolished the transactivation by ER. This finding is very interesting, as this AT-rich site is recognized by the nuclear matrix protein/scaffold attachment factor HET/SAF-B, which interacts with ER and represses its transcription of this promoter (30). Therefore, these findings could be explained by one of two possibilities. Firstly, it is possible that when Brn-3b proteins are bound to the site between the two 1/2 EREs, its interaction with the ER facilitates the strong binding of the ER complex to the promoter and enhances transcriptional activation. Because Brn-3b can bind to ER, off as well as on DNA (17), mutation of the Brn-3 site will result in loss of Brn-3b binding to this site, but it will continue to bind to ER and remove it from the ERE in the promoter. This will compromise ER complex formation on the promoter with consequent loss of transactivation. Alternatively, it is possible that the binding of Brn-3b to this site prevents the binding of repressor protein, HET protein, which also binds to this sequence (30). Loss of Brn-3b binding to this site may facilitate the binding of HET factor, which then interacts with and inhibits ER-mediated transcription (30, 36). Therefore, the balance of proteins, such as Brn-3b or HET/SAF-B, which are coexpressed with the ER in breast cancer cells, will serve to regulate the expression of factors, such as HSP-27, which can act to modify cellular behavior.
The loss of ER-mediated transactivation on targeting of Brn-3b using RNAi would support a significant role for Brn-3b in cooperation with the ER for maximal stimulation of HSP-27 promoter, thus supporting the hypothesis that proposes that Brn-3b binds to and facilitates formation of the ER complex on the HSP-27 promoter and as such is required for maximal stimulation of the promoter.
It is interesting that overexpression of HSP-27 in breast cancer cells gives rise to similar changes observed with Brn-3b overexpression in breast cancer cell line, MCF-7. Thus, independent studies have shown that elevation of Brn-3b or HSP-27 results in increased anchorage-dependent growth and proliferation as well as anchorage-independent colony formation (6, 10, 16, 37). Moreover, the ability of HSP-27 to confer resistance to chemotherapeutic drugs in cancer cells (4, 6) is paralleled by our observation that high levels of Brn-3b protein in neuroblastoma cells result in failure to respond to a growth inhibitory stimulus (16). Thus, many of the effects associated with high HSP-27 are also seen with elevated Brn-3b, suggesting a similar pathway by which the cells are affected. The correlation of Brn-3b with levels of HSP-27 in breast cancer cell lines and breast biopsies together with the regulation of the HSP-27 promoter by Brn-3b either alone or when coexpressed and interacting with ER would suggest that the regulation of HSP-27 by Brn-3b is important in determining the downstream effects observed in cancer cells and may thus represent one mechanism by which this transcription may contribute to changes observed in breast cancer cells.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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5 Samady et al., submitted for publication. 
Received 8/ 9/04. Revised 12/ 8/04. Accepted 2/11/05.
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0.0002, compared with control, Student's t test. Values have been equalized for the Renilla luciferase control, which was included in all cotransfections to control for variation in transfection efficiency. Percentage of the empty LTR vector control is set at 100%. Columns, mean of at least three independent experiments; bars, SE. B, to test whether Brn-3b cooperated with ER on the HSP-27 promoter, similar cotransfection studies with the HSP-27 promoter construct were carried out with either Brn-3b or ER alone or in combination in MCF-7 cell line (as above). The ER alone could stimulate promoter activity to a higher level compared with Brn-3b(s) or Brn-3b(l), but addition of Brn-3b with the ER resulted in a significant enhancement of promoter activity (P 
