Brn-3b transcription factor enhances proliferation of neuroblastoma (NB) and breast cancer cell lines in vitro and increases the rate and size of in vivo tumour growth, whereas reducing Brn-3b slows growth, both in vitro and in vivo. Brn-3b is elevated in >65% of breast cancer biopsies, and here we demonstrate that Brn-3b is also elevated in NB tumours. We show a significant correlation between Brn-3b and cyclin D1 (CD1) in breast cancers and NB tumours and cell lines. Brn-3b directly transactivates the CD1 promoter in co-transfection experiments, whereas electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrate that Brn-3b protein binds to an octamer sequence located in the proximal CD1 promoter. Site-directed mutagenesis of this sequence resulted in loss of transactivation of the CD1 promoter by Brn-3b. Thus, Brn-3b may act to alter growth properties of breast cancer and NB cells by enhancing CD1 expression in these cells.
Brn-3b transcription factor was originally isolated from neuroblastoma (NB)-derived ND7 cell line on the basis of homology within the conserved Pit-Oct-Unc (POU) domain of the related Brn-3a protein (Lillycrop et al., 1992). Brn-3b is expressed in neurons of the developing and adult nervous system (Xiang et al., 1993; Turner et al., 1994; Xiang, 1998) but also in non-neuronal tissues including breast epithelium (Budhram-Mahadeo et al., 2001; Budhram-Mahadeo and Latchman, 2006). Brn-3b proteins have complex functions that depend on which cell types express them. For instance, in the retina, Brn-3b is expressed in the ventricular zone and in retinal ganglion cells (RGC) (Xiang et al., 1993, 1996) and is essential for normal retinal development since Brn-3b ‘knock-out’ (KO) mice are blind (Erkman et al., 1996; Gan et al., 1996). This results from inappropriate differentiation/axonal outgrowth and death within the RGC layer and ventricular zone during late embryogenesis and post-natally (Xiang, 1998).
In contrast to its effects in the retina, elevated expression of Brn-3b in cancer cells is strongly associated with increased proliferation and altered growth. Brn-3b expression in proliferating NB cell lines is decreased if cells undergo differentiation by serum withdrawal or upon stimulation with retinoic acid (RA) (Budhram-Mahadeo et al., 1995; Smith and Latchman, 1996). Furthermore, forced overexpression of Brn-3b protein in breast cancer and NB cells increases proliferation in vitro and enhances in vivo tumour growth in xenograft models, whereas reducing Brn-3b (by antisense) decreases proliferation in vitro and result in smaller, slower growing tumours in vivo (Dennis et al., 2001; Irshad et al., 2004). Brn-3b also confers resistance to growth inhibitory stimulus, RA, and increases migratory potential of NB cells (Irshad et al., 2004). Brn-3b is elevated in >65% of breast cancers (Budhram-Mahadeo et al., 1999), but its expression in primary NBs has not so far been studied.
As a transcription factor, Brn-3b protein regulates the expression of critical genes that control different processes; for example, in neuronal cells, Brn-3b repress genes associated with differentiation, namely, α-internexin, SNAP25 and synaptophysin (Latchman, 1998a), whereas in breast cancer cells, it represses BRCA1 associated with cell cycle arrest (Budhram-Mahadeo et al., 1999). In contrast, Brn-3b activates heat shock protein 27 (Hsp27), associated with invasiveness and drug resistance in breast cancers (Lee et al., 2005) and the cyclin-dependent kinase, CDK4, required for cell cycle progression (Samady et al., 2004).
CDK4 protein controls G1 transition in cells as they progress through the cell cycle (Arnold et al., 1991) by associating with regulatory partners such as cyclin D1 (CD1) (also referered to as PRAD1, CCND1) (Motokura et al., 1991) to phosphorylate specific substrates such as retinoblastoma protein (Israels and Israels, 2000). However, whereas CDK4 is inhibited by its association with inhibitory protein such as p21cip1/waf1, the regulatory cyclin subunits are regulated at the level of expression and by protein stability as cells progress through the cell cycle (Guo et al., 2005). Therefore, factors that control expression of CD1 will play an important role in cell cycle progression, particularly through the G1 phase.
Like Brn-3b, CD1 is critical for normal retinal development (Fantl et al., 1995; Sicinski et al., 1995) since CD1 KO mice suffer loss of retinal cells with resultant blindness (Sicinski et al., 1995). More importantly, a 2.5-fold decrease in CD1 is seen in retinas of Brn-3b KO mutants compared with wild-type (WT) littermates in microarray analysis (Mu et al., 2001). Similar to Brn-3b, CD1 also increases proliferation of breast cancer cells (Zwijsen et al., 1996) and is elevated in many breast cancers (Barnes and Gillett, 1998; Budhram-Mahadeo et al., 1999). Furthermore, transgenic mice that are engineered to overexpress CD1 protein in the breast, develop tumours in the mammary gland (Wang et al., 1994).
In this study, we show correlation between Brn-3b with CD1 in breast tumours and NBs and demonstrate that Brn-3b proteins could directly transactivate CD1 promoter.
Results and discussion
Brn-3b mRNA is expressed at high levels in biopsies taken from patients with NB tumours
Because Brn-3b could significantly enhance growth and migratory potential, while conferring resistance to growth inhibitory stimulus in NB cell lines (Irshad et al., 2004), we tested whether Brn-3b was elevated in NB tumour biopsies. Quantitative reverse-transcription–PCR (qRT–PCR) was used to measure total Brn-3b mRNA from cDNA (prepared from RNA obtained from NB biopsies). The related Brn-3a POU transcription factor (associated with differentiation in NB cell lines) was also measured. Values were adjusted using housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and differences were expressed as fold changes in Brn-3b or Brn-3a, compared to levels found in control (human SHSY5Y cells – set at 1). Figure 1a shows high levels of Brn-3b mRNA in many tumour samples, with ∼75% showing elevated Brn-3b (over 100-fold higher) compared with levels found in control. In contrast, majority of these tumour samples had very low levels of the mRNA encoding the related but antagonistic Brn-3a. This opposite expression of Brn-3b and Brn-3a in NBs reflects their unique effects in NB cell lines. Thus, unlike the growth-promoting effects of Brn-3b, high levels of Brn-3a lead to differentiation of NB cells as a consequence of differential effects on specific target genes (Smith and Latchman, 1996; Latchman, 1998b). In this regard, elevated Brn-3b and low levels of Brn-3a in NB biopsies are also reflected in their opposite and antagonistic effects in NB cell lines. Taken together with previous results in NB cell lines, it is likely that elevated Brn-3b in NBs will play an important role in regulating the growth and behaviour of these tumours.
Further analysis of these NBs, according to tumour stages (using International Neuroblastoma Staging System (INSS)), (Brodeur, 2003) showed highest levels of Brn-3b in stage 3 tumours (Figure 1b) with a median of 1500 compared with less aggressive stages 1/2 (median ∼222) or 4 (median ∼344.5). Interestingly, higher stage 4 tumours appeared to express lower Brn-3b (median ∼362) than stage 3 tumours.
Aggressive stage 4 NBs often have MYCN amplification, so Brn-3b levels were further analysed using MYCN status of the tumours. Figure 1c shows lower Brn-3b levels in stage 4 tumours with MYCN amplification compared with those with no MYCN amplification (NA) (Figure 1c) (mean values of 12 NB samples with MYCN amplification ∼362-fold increase compared with mean of ∼548 for similar numbers of NB tumours with normal copies of MYCN). However, because of the wide variation in Brn-3b levels in these tumours and small sample size, this is not statistically significant (P=0.2), and larger sample numbers will be needed to confirm these findings.
These results suggest that Brn-3b is significantly elevated in many NB tumours, particularly in stage 3 and 4 tumours that do not have MYCN amplification.
CD1 correlates with Brn-3b in cell lines and tumour samples
Since Brn-3b TF enhances cell proliferation whereas loss of Brn-3b slowed cell growth (Dennis et al., 2001; Irshad et al., 2004), we measured CD1 protein (associated with proliferation) in stably transfected NB cells that overexpressed Brn-3b (Brn-3b+) or had reduced Brn-3b protein (3b antisense) (Irshad et al., 2004) compared with control cells. Figures 2A(i) and (ii) show significantly higher levels of endogenous CD1 protein in Brn-3b (+) cells compared with controls (P-value<0.05), whereas Brn-3b antisense cells had reduced CD1 levels. This decrease in CD1 upon loss of Brn-3b was also observed in MCF7 breast cancer cells that stably expressed antisense to Brn-3b (Figure 2A(iii)). Thus, altering Brn-3b protein levels in NB cells or breast cancer cells were sufficient to change the growth of these cells and this was paralleled by changes in CD1 expression.
We next quantified CD1 in tumours with known Brn-3b levels using RNA obtained from 48 NBs (Figure 1) and 42 (previously analysed) breast cancer biopsies (Budhram-Mahadeo et al., 1999; Samady et al., 2004; Lee et al., 2005). Figure 2B(i) shows good correlation of Brn-3b with CD1 mRNA in >50% of NB samples (R=0.55), which was specific to CD1 since there was poor correlation between cyclin E and Brn-3b in these tumours (R=0.0002) (Figure 2B(ii)). Similar analysis of 42 breast cancer biopsies demonstrated an even stronger correlation (R=0.85) between Brn-3b and CD1 (Figure 2C), confirming a close association between Brn-3b and CD1 in tumour samples and cell lines expressing different levels of Brn-3b protein.
Transactivation of CD1 promoter by Brn-3b
We next tested whether Brn-3b could directly regulate CD1 expression by undertaking transient transfection studies in which CD1 promoter reporter plasmid (CD1-848) was co-transfected with Brn-3b expression vector (in which long terminal repeat (LTR) drives expression of Brn-3b cDNA) into ND7 cells. Deletion constructs, CD1-567 or CD1-69 (Sabbah et al., 1999; Redeuilh et al., 2002) (Figure 3a) were used to map the region of CD1 promoter required for Brn-3b transactivation. TK-renilla reporter (included in all transfections) provided an internal control for variability in transfection efficiencies.
Figure 3b(i) shows that Brn-3b transactivated the longest CD1 promoter (CD1-848) by 8- to 10-fold, compared with vector control, LTR (P<0.005). Similar transactivation was observed when deletion construct, CD1-573, was co-transfected with Brn-3b but the shortest promoter construct, CD1-69, was not transactivated by Brn-3b, although its basal activity was comparable to the longest promoter CD1-848 (relative luciferase assay unit (RLU) following transfection of each promoter alone) (Figure 3b(ii)). Figure 3b(iii) shows increased Brn-3b proteins in cells following transfection with Brn-3b expression vector. These results suggest that CD1-69 promoter construct contains the elements required for assembly of the intact basal transcription machinery but the regulatory elements required for Brn-3b to transactivate this promoter reside within sequence −567 and −69 of the promoter and are lost in CD1-69 construct.
We also tested whether Brn-3b could transactivate full-length CD1 promoter, CD1-848, in MCF7 breast cancer cell lines, using similar experiments. Figure 3c shows that Brn-3b strongly transactivated the CD1 promoter in breast cancer cells also.
The ability of Brn-3b to transactivate CD1 promoter in NB and breast cancer cells suggests that the correlation between these two factors in tumour samples may result from direct regulation of CD1 by Brn-3b.
Brn-3b binds directly to an octamer site in the proximal CD1 promoter
Analysis of the sequence between −567 and −69 of the CD1 promoter for putative transcription factor binding sites, using MatInspector (http://www.genomatix.de/), revealed two A/T-rich sequences between positions −248 and −228 (Figure 4a(i)), which had homology to octamer sites recognized by these POU proteins. To test whether Brn-3b binds to this region of the CD1 promoter, electrophoretic mobility shift assay (EMSA) was performed using 32P-radiolabeled oligonucleotides probes (corresponding to this sequence). Figure 4a(ii) shows retarded protein/DNA complexes formed when the probe was incubated with Brn-3b (+) protein extracts (indicated by *) but not present when using SAOS2 cell extracts (lacking endogenous Brn-3b). These bands were specifically competed upon addition of unlabeled Brn-3 binding site (specific competitor) but not by nonspecific oligonucleotide (NS). Addition of Brn-3b antibodies (but not anti-actin antibodies) resulted in supershift of the protein/DNA complexes (indicated by double *). These results confirm that Brn-3b proteins bind to this region of CD1 promoter, specifically and strongly, and the presence of complexes with different mobilities suggests that Brn-3b may bind to DNA in different configuration (for example, monomer or hetero-/homo-dimer).
We next tested which of two A/T sites, found within this region, was required for binding of Brn-3b to CD1 promoter by introducing mutations into each site (mutant 1 and mutant 2) (Figure 4b(i)). 32P-labeled mutant oligonucleotide probes were used in EMSA and compared with WT probe. Figure 4b(ii) shows the expected complexes following incubation of cell extracts with WT probe but no specific bands were seen following incubation of cell extract with labeled mutant 1 probe (TTT> >GGG at positions 240–237) and no changes occur upon addition of specific or nonspecific competitor. In contrast, incubation of cell extract with mutant 2 probe (TTT> >TGG at positions 246–243) resulted in a similar pattern of complexes seen with WT probe, that is, formation of complexes that were competed by specific competitor but not nonspecific competitor. Thus, TTT at positions 240–237 appears to be critical for binding of Brn-3b to the CD1 promoter and suggests that sequence 5′-IndexTermATTTCTAT-3′ found between −248 and −228 (from transcriptional start site), in CD1 promoter is likely to be the octamer site bound by Brn-3b.
Site-directed mutagenesis of octamer site within −248 to −228 in CD1 promoter prevented transactivation by Brn-3b
To test whether this sequence was essential for Brn-3b-mediated activation of CD1 promoter, site-directed mutagenesis was used to introduce mutations into three bases within this site in the longest CD1-848 promoter reporter (Figure 4c(i)). The resultant mutated promoter was co-transfected with Brn-3b into ND7 cells and promoter activity compared with WT CD1 promoter activity in the same experiment. Figure 4c(ii) shows that Brn-3b transactivated WT CD1 promoter (15- to 20-fold), but this effect was significantly reduced on the mutated promoter (three- to five-fold). Thus, we have identified the sequence that is necessary for effective Brn-3b-mediated transactivation of the CD1 promoter.
Brn-3b is bound to CD1 promoter in intact cells
Chromatin immunoprecipitation (ChIP) assays were undertaken to test whether Brn-3b is bound to the CD1 promoter in vivo, in intact cells (Lee et al., 2005). Proteins bound to chromatin in MCF7 cells were immunoprecipitated, using Brn-3b antibody or control actin antibody. Figure 4d shows that PCRs (with primers to amplify CD1 promoter flanking the octamer site), using ChIP DNA immunoprecipitated with Brn-3b antibody, give rise to a product that corresponded to the size seen with the input sample (positive control), but this was not observed when using the ChIP DNA obtained with anti-actin antibody. The increased intensity of the amplified product when using DNA obtained following ChIP from Brn-3b overexpressing cells (Brn-3b+) (Figure 4d(i)) shows that more CD1 promoter was immunoprecipitated in presence of higher levels of Brn-3b. Brn-3b is specifically bound to the proximal promoter because amplification of the distal region of the CD1 promoter (∼2.7 kb from binding site), using the same chromatin DNA (immunoprecipitated with Brn-3b Ab), did not give rise to the product which was seen when using the input (positive control) (Figure 4d(ii)). These results confirm that Brn-3b binds directly to proximal CD1 promoter in intact cells, thus facilitating promoter activation.
Our results therefore suggest that the correlation between Brn-3b and CD1 in breast cancers and NBs is likely to arise from the ability of Brn-3b to directly regulate CD1 expression. This relationship also exist in non-transformed cells since Brn-3b and CD1 have overlapping expression during retinal development and loss of Brn-3b result in reduction of CD1 in Brn-3b KO retinas (Mu et al., 2001). Interestingly, also, both CD1 and Brn-3b mutants are blind (Sicinski et al., 1995; Gan et al., 1996). Thus, expression of Brn-3b and CD1 are closely associated in different tissues, suggesting that Brn-3b-mediated transactivation of CD1 expression is important in distinct cell types.
The proliferative effects of CD1 relate to its control of G1 transition in specific cell types (Arnold et al., 1991), and although it functions primarily by forming active complexes with CDK4 (Israels and Israels, 2000), CD1 can also act via mechanisms that are independent of CDK4 (Zwijsen et al., 1997; Knudsen et al., 1999; Bienvenu et al., 2001). Elevated CD1 in tumours can be achieved through different mechanisms including deregulated gene expression, mutations or amplifications (Weinberg, 1995). In NB cell lines and tumours, increased CD1 is not associated with mutations or amplifications (Molenaar et al., 2003) and are likely to result from deregulation of pathways that control CD1 expression in these cells. Given that elevated Brn-3b can significantly enhance growth and proliferation in breast cancer and NB cells and reducing endogenous expression slows their growth, it is likely that the Brn-3b transcription factor mediates its effects, in part, by transactivating effectors of the cell cycle such as CD1 and CDK4 (Samady et al., 2004), thus allowing cell cycle progression and promoting cellular proliferation.
Materials and methods
Reporter constructs containing cyclin D1 promoter (CD1-848) or deletion constructs (CD1-567 and CD1-69) cloned into pGL2 luciferase vector were kind gifts from Dr Gerard Redeuilh (Saint-Antoine Hospital, Paris, France). Brn-3b expression vectors contain cDNA encoding either Brn-3b(s) or genomic DNA capable of encoding both Brn-3b(l) and Brn-3b(s) (Lee et al., 2005). pRL-TK (Promega, Southampton, UK) containing Renilla Luciferase reporter gene under control of herpes simplex virus-thymidine kinase promoter was used to control for transfection efficiency.
α-Brn-3b antibody (goat polyclonal), α-cyclin D1 mAb (DCS-6) and α-actin (I-19) (goat polyclonal) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Secondary antibodies were obtained from Dako (Cambridgeshire, UK); used at 1:2000–1:3000).
RNAs from neuroblastomas were obtained from the Italian Neuroblastoma Society (Genoa, Italy). Neuroblastoma biopsies were provided by Pathology Department, Great Ormond Street Hospital (London, UK) or UKCCSG, UK. RNA from breast cancer biopsies were provided by Candis Tissue Bank (Liverpool, UK), while breast cancer biopsies were provided by CR-UK Hedley Atkins Breast Pathology Laboratory (Guy's Hospital, London, UK).
Cell culture, transient transfections, luciferase assay
Neuroblastoma ND7 cell line (Budhram-Mahadeo et al., 1994a) and MCF7 breast cancer cell lines were grown in the appropriate growth medium (Lillycrop et al., 1992; Budhram-Mahadeo et al., 1994a; Samady et al., 2004) until subconfluent, then plated for experiments. Transient transfections were carried out with the specified reporter/expression vectors in the ND7 cell line using Fugene (Roche, Welwyn Garden City, UK) as described (Hudson et al., 2005). The renilla reporter was included in all transfections to provide an internal control for variation in transfection efficiencies. Cells were harvested after 48 h in passive lysis buffer and luciferase reporter analysis undertaken using the dual luciferase assay system (Promega) and TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA) as described (Lee et al., 2005).
RNA extraction and cDNA synthesis
Cells were harvested in TRIZOL reagent (Invitrogen, Paisley, UK) and processed according to the manufacturer's protocol. For RNA isolation from tumour biopsies, tissues were homogenized in liquid nitrogen, resuspended in Tirzol and then processed according to the manufacturer's protocol. DNAse1 (RNAse free) treatment was used to remove contaminating genomic DNA (Amersham Pharmacia Biotech, Buckinghamshire, UK) followed by phenol/chloroform extraction and ethanol precipitation of RNA. Total RNA (0.5–1 μg) was used for cDNA synthesis in 20–50 μl reaction using SUPERSCRIPT II, RNase H-Reverse Transcriptase (Invitrogen). This cDNA (1–2 μl) was used in subsequent qRT–PCR for each target gene to be studied or the housekeeping gene, GAPDH.
Quantitative real-time RT–PCR
TaqMan Gene Expression Assay from Applied Biosystems (Warrington, UK) were used for amplification and quantification of human cyclin D1 (hCG2016647) and for human GAPDH (99999905). Total Brn-3b transcripts were measured using qRT–PCR, in which custom probe and primers were designed using ABI assay-by-design file builder programme (http://www.appliedbiosystems.com). QuantiTect Probe PCR Kit used for the qRT–PCR was obtained from Qiagen (West Sussex, UK). Reactions were carried out using the DNA Engine Opticon (MJ Research, GRI Braintree, Essex, UK) and conditions were as stipulated for use with TaqMan Gene Expression Assay. The comparative Ct method was used to determine changes in relative levels of different genes (Vandesompele et al., 2002). The Ct values of samples of interest were compared with the calibrator, for example, control untreated cell line (ND7) or untreated SHSY5Y human neuroblastoma cells, or MCF7 breast cancer cells were used for tumour samples and normalized to endogenous GAPDH housekeeping gene. For standard curve method, a known amount of each target was prepared as a series of serial dilution and once tested to show that a linear standard curve was produced; aliquots of the each standard were frozen so that the same sets of standards was used for all subsequent experiment to ensure reproducibility in the studies.
Electrophoretic mobility shift assay
EMSA was performed as described previously (Lee et al., 2005). Double-stranded oligonucleotides (containing the putative Brn-3 site in the cyclin D1 promoter) comprised of sequence 5′-IndexTermCGATTTGCATTTCTATGAAAA-3′ (or mutant oligonucleotides, see Figure 4b) were end-labeled with T4 kinase. Labeled probe (1 ng) was incubated with cellular extract prepared either from IMR-32 neuroblastoma cells overexpressing Brn-3b protein or from SAOS2 cells (that do not express Brn-3b) under conditions described (Lee et al., 2005). The protein–DNA complex was resolved from the free probe by electrophoresis and detected by autoradiography.
Chromatin immunoprecipitation assay
This technique was carried out as described by Lee et al. (2005) using Brn-3b overexpressing MCF7 cells and antibody to either Brn-3b (goat pAb); or actin pAb. Proximal region of cyclin D1 promoter (containing Brn-3 site) was amplified using primers CD1F1: 5′-IndexTermGACCCACTCGAGGCGGAC-3′ and CD1R: 5′-IndexTermCGCCAAACGCCGGGAGCA-3′, whereas distal promoter region was amplified using primers CD1f-1.5Kb: 5′-IndexTermCAATGACCCTCAAAAGCCCAG-3′ and CD1r-1.5Kb: 5′-IndexTermAACCGGGCTTGCAATTTTGCG-3′. Standard conditions were used for PCR (2.5 mM MgCl2 with cycling parameters of one cycle at 94°C for 15 min; 40 cycles of 95°C for 30 s; 58°C for 30 s and 72°C for 30 s followed by 72oC for 5 min. PCR products were resolved on 2.5% agarose/TBE gel.
Data points from independent experiments were compared using Student's t-test analysis and the level of significance of the test is given as P-value in the text. Regression analysis was undertaken using SigmaPlot 2001 programme to establish correlation of different factors.
electrophoretic mobility shift assay
long terminal repeat
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We thank Dr Gerard Redeuilh (Paris, France) for cyclin D1 reporter constructs; Dr J Anderson and Dr P Brock (Great Ormond Street Hospital (GOSH), London) for support and discussions; Italian Neuroblastoma Society; Pathology Department (GOSH, London) and UKCCSG, UK for NB RNA or biopsies; Dr D' Arrigo (Guy's Hospital, London) for breast cancer biopsies and Candis Tissue Bank (Liverpool, UK) for RNA from breast cancer. This work was supported by Child Health Research Action Trust (CHRAT); Breast Cancer Campaign (BCC) UK; Association for International Cancer Research (AICR), UK.
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