1 Laboratoire d'OncogénétiqueINSERM E0017, Centre René Huguenin, St-Cloud, France and 2 Laboratoire de Génétique MoléculaireUPRES EA 3618, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris V, Paris, France
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Abstract |
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Abbreviations: ER, estrogen receptor alpha; ERß, estrogen receptor beta; MMP, matrix metalloproteinases; RTPCR, reverse transcriptasepolymerase chain reaction; SD, standard deviation; TBP, TATA box-binding protein
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Introduction |
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PEA3 expression is mainly regulated by two distinct Ras-dependent mitogen activated protein kinase (MAPK) pathways (13). These MAPK cascades could be activated by upstream molecules including receptor tyrosine kinase such as ERBB2 receptor (14) or growth factors such as heregulin (15) and hepatocyte growth factor (16). It is noteworthy that the relationship between PEA3 and ERBB2 is unclear. Xing et al. (12) suggest that PEA3 is able to repress ERBB2 gene expression while other authors suggest that up-regulation of ERBB2, via activation of MAPK cascades, results in increased PEA3 transcriptional activity (14). In consequence, the function of PEA3 (oncogene or tumor-suppressor gene) should be clarified before the use of new PEA3-based therapies. Indeed, a phase II study has already been started to evaluate the therapeutic efficiency and tumor-suppression mechanisms of PEA3 (17).
A recent publication also suggested the involvement of epigenetic mechanisms (DNA methylation) in the regulation of PEA3 gene expression (18). These authors have analyzed 10% of all mouse genes by transcription profiling from conditional DNMT1-deficient primary mouse fibroblasts to identify genes induced by hypomethylation. These authors identified PEA3 gene as a major gene up-regulated by DNA hypomethylation via DNMT1 under-expression in the mouse cells.
The role of PEA3 in human breast cancer is poorly documented, and even less is known about the clinical significance of PEA3 expression in this setting (19,20). The few available data on PEA3 expression in breast cancer have been obtained using breast cancer cell lines (21) and mouse mammary tumors (22,23). Kaya et al. (24) showed that transfection of the non-invasive human breast cancer cell line MCF-7 with PEA3 gene results in induction of invasive and motile activities, accompanied by an up-regulation of MMP9. PEA3 is over-expressed in mammary tumors of MMTV-ERBB2 transgenic mice (22). Expression of a dominant-negative PEA3 transgene in mammary epithelial cells of MMTV-ERBB2 transgenic mice dramatically delayed the onset of mammary tumors and reduced the number and size of such tumors in individual mice (20). Taken together, these findings suggest a role of this transcription factor in breast cancer.
Only two clinical studies of women series with breast tumors examined the possible association between PEA3 expression and classical clinical and pathological parameters, including patient outcome (25,26). Benz et al. (25) examined PEA3 expression by in situ hybridization in a series of 74 human breast tumors without outcome. This study revealed that (i) PEA3 is over-expressed (>2-fold) in the epithelial compartment of 76% of these breast tumors, (ii) PEA3 and ERBB2 are coordinately up-regulated, (iii) PEA3 over-expression does not correlate with nuclear grade, estrogen receptor and S-phase fraction, and does not result from amplification of the PEA3 gene located in 17q22. The second study, realized at the protein level by immunohistochemistry, suggested that PEA3-positive status detected in 42% of 89 patients could be a factor of good prognostic significance (26).
PEA3 is supposed to play an important role in breast cancer invasiveness/metastasis through transcription of numerous metastasis-related genes. However, no data are available on the expression pattern of both PEA3 gene and genes involved in the PEA3 signaling network in a series of human breast tumors.
Here, using real-time quantitative reverse transcriptase polymerase chain reaction (RTPCR) assay, we quantified PEA3 mRNA expression in a series of 130 patients with unilateral invasive primary breast tumors and known long-term outcome. We then sought links between the PEA3 mRNA expression pattern and classical clinical, pathological and molecular parameters, including patient outcome.
We also examined the possible relationship between the expression of PEA3 and those of genes coding for (i) molecules putatively involved in upstream steps of PEA3 pathways including growth factors (EGF, AREG, EREG, NRG1, NRG2, NRG3, HGF) and their receptors (ERBB1, ERBB2, ERBB3, ERBB4), (ii) molecules putatively involved in downstream steps of PEA3 pathways including various matrix proteases (UPA, PLAT, MMP2, MMP7, MMP9, MMP14), their inhibitors (PAI1, PAI2, TIMP1, TIMP2, TIMP3, TIMP4) and the genes GJB2 (Connexin 26) and PLAUR (UPA receptor) and (iii) DNA methyltransferases (DNMT1, DNMT3A, DNMT3B) as well as genes (GRO1, JUNB) highly co-up-regulated with PEA3 gene by DNA hypomethylation in conditional DNMT1-deficient primary mouse fibroblasts (18).
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Materials and methods |
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The patients (mean age 58.2 years, range 3491) met the following criteria: primary unilateral non-metastatic breast carcinoma on which complete clinical, histological and biological data were available; and no radiotherapy or chemotherapy before surgery. The histological type of each tumor, and the number of positive axillary nodes, were established at the time of surgery. The malignancy of infiltrating carcinomas was scored according to Bloom and Richardson's histoprognostic system. Estrogen receptor alpha (ER) status was determined by real-time quantitative RTPCR assay (27). The main classical prognostic factors of this breast tumors series have been reported previously (27).
The median follow-up was 8.1 years (range 1.015.9). Forty-seven patients relapsed (the distribution of first relapse events was as follows: 13 local and/or regional recurrences, 30 metastases, and four of both).
We also analyzed eight breast tumor cell lines obtained from the American Tissue Type Culture Collection (SK-BR-3, T-47D, BT-20, HBL-100, ZR-75-1, MDA-231, MDA-361 and MCF7).
Specimens of adjacent normal breast tissue from five of the breast cancer patients, and normal breast tissue from four women undergoing cosmetic breast surgery, were used as sources of normal RNA.
Real-time RTPCR
Theoretical basis
Quantitative values are obtained from the cycle number (Ct value) at which the increase in fluorescent signal associated with an exponential growth of PCR products starts to be detected by the laser detector of the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA) using the PE Biosystems (Foster City, CA) analysis software according to the manufacturer's manuals.
The precise amount of total RNA added to each reaction mix (based on optical density) and its quality (i.e. lack of extensive degradation) are both difficult to assess. We therefore also quantified transcripts of the gene coding for the TATA box-binding protein (TBP) (a component of the DNA-binding protein complex TFIID) as the endogenous RNA control, and each sample was normalized on the basis of its TBP content.
The relative target gene expression level was also normalized to a calibrator, or 1x sample, consisting of a pool of normal breast tissue specimens (quantification of PEA3 gene) or of the breast tumor tissue sample among the tested series which contained the smallest amount of target gene mRNA (quantification of other target genes).
Final results, expressed as N-fold differences in target gene expression relative to the TBP gene and the calibrator, termed NPEA3, were determined as follows:
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Primers and PCR consumables
Primers for the TBP and target genes were chosen with the assistance of the computer programs Oligo 5.0 (National Biosciences, Plymouth, MN). We conducted BLASTN searches against dbEST, htgs and nr (the non-redundant set of the GenBank, EMBL and DDBJ database sequences) to confirm the total gene specificity of the nucleotide sequences chosen for the primers, and the absence of DNA polymorphisms. The primer pairs for PEA3 were selected to be unique when compared with the sequences of the closely related ER81/ETV1 and ERM/ETV5 genes. The nucleotide sequences of the primers used for PCR amplification of PEA3, TBP, MKI67 and other target genes are reported in Table I. To avoid amplification of contaminating genomic DNA, one of the two primers was placed in a different exon. For example, the upper primer of TBP was placed at the junction between exons 5 and 6, whereas the lower primer was placed in exon 6. Agarose gel electrophoresis allowed us to verify the specificity of PCR amplicons. Moreover, for each primer pair, we did no-template control and no-reverse transcriptase control (RT negatively) assays, which produced negligible signal detection (usually >40 Ct in value), suggesting that primerdimer formation and genomic DNA contamination effects were negligible.
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cDNA synthesis
RNA was reverse transcribed in a final volume of 20 µl containing 1x RT buffer (500 µM each dNTP, 3 mM MgCl2, 75 mM KCl, 50 mM TrisHCl pH 8.3), 20 U RNasin Ribonuclease inhibitor (Promega, Madison, WI), 10 mM dithiothreitol, 100 U Superscript II RNase H-reverse transcriptase (Gibco BRL, Gaithersburg, MD), 1.5 µM random hexamers (Pharmacia, Uppsala, Sweden) and 1 µg of total RNA. The samples were incubated at 20°C for 10 min and 42°C for 30 min, and reverse transcriptase was inactivated by heating at 99°C for 5 min and cooling at 5°C for 5 min.
PCR amplification
All PCR reactions were performed using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems). PCR was performed using the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems). The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min and 50 cycles at 95°C for 15 s and 65°C for 1 min.
Statistical analysis
Relapse-free survival was determined as the interval between diagnosis and detection of the first relapse (local and/or regional recurrences, and/or metastases).
Clinical and pathological parameters were compared using the 2 test. Spearman rank correlation test was used to study the association between continuous variables.
Survival distributions were estimated by the KaplanMeier method (28), and the significance of differences between survival rates was ascertained using the log-rank test (29).
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Results |
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PEA3 mRNA expression in breast tumors
NPEA3 values in the 130 breast tumor RNA samples ranged from 0.02 to 15.7, i.e. nearly 3 orders of magnitude. Compared with normal breast tissues, 30 tumors (23.1%) under-expressed PEA3 mRNA and 18 tumors (13.8%) over-expressed it.
PEA3 expression was also measured in eight breast tumor cell lines: BT-20 and MDA-231 showed PEA3 over-expression (NPEA3 values of 9.5 and 5.2, respectively), SK-BR-3, HBL-100 and ZR-75-1 showed normal PEA3 expression (NPEA3 values of 1.45, 1.30 and 0.98, respectively) and MCF7, MDA-361 and T-47D showed PEA3 under-expression (NPEA3 values of 0.15, 0.22 and 0.12, respectively).
The NPEA3 values (calculated as described in Materials and methods) were based on the amount of the PEA3 messenger relative to the TBP endogenous control to normalize the amount and quality of total RNA. TBP expression remains stable between the normal breast tissue samples and the breast tumor samples when it is normalized to a second endogenous RNA control, the RPLP0 gene (also known as 36B4), coding for the human acidic ribosomal phosphoprotein P0. In consequence, we obtained the same results concerning PEA3 gene expression in our breast tumor series when its expression is normalized to this second endogenous RNA control (RPLP0 gene).
Relationship between PEA3 mRNA levels and clinical and pathological parameters
We sought links between PEA3 mRNA expression status and standard clinical and pathological factors in breast cancer (Table II). Links (or trends) were found between PEA3 gene status and ScarffBloomRichardson (SBR) histopathological grade status (P = 0.018) and lymph node status (P = 0.075).
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Relationship between PEA3 mRNA levels and molecular parameters
The 130 tumors studied here for PEA3 mRNA expression had been tested previously for ER, ERß and ERBB2 mRNA expression (Table III) (27,30). We observed a negative relationship between PEA3 and ER
mRNA levels (r = -0.277, P = 0.0016) and a positive relationship between PEA3 and ERBB2 mRNA levels (r = +0.175, P = 0.045). No link was observed between PEA3 and ERß mRNA levels.
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Relationship between PEA3 mRNA levels and that of various putative genes implicated in the PEA3 pathway dysregulation in breast cancer
The possible relationship between the expression of PEA3 and of 30 candidate genes selected to be implicated in PEA3 pathway dysregulation by various studies, were analyzed in 11 PEA3-under-expressing and 11 PEA3-over-expressing breast tumors. These genes encode various growth factors and their receptors (EGF, AREG, EREG, NRG1, NRG2, NRG3, ERBB1, ERBB2, ERBB3, ERBB4, HGF), extracellular matrix and cell-surface molecules (UPA, PLAT, MMP2, MMP7, MMP9, MMP14, PAI1, PAI2, TIMP1, TIMP2, TIMP3, TIMP4, GJB2, PLAUR), DNA methyltransferases (DNMT1, DNMT3A, DNMT3B), as well as genes putatively up-regulated by hypomethylation (GRO1, JUNB).
To correct for variations in the amounts of RNA, the mRNA levels of PEA3 and of these 30 genes were normalized using the TBP endogenous control gene, which is a non proliferation-associated gene. As the PEA3 gene expression is proliferation dependent, the PEA3 and the 30 genes were also normalized using the MKI67 gene. The possible relation between the 30 genes and ER gene expression was also examined in the same subset of 22 tumors. The results of these analyses are summarized in Table IV.
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Among these six genes, EREG, NRG3, ERBB1 and MMP7 also correlated negatively with ER gene expression (Table IV, column ER
). The associations between PEA3 and these four genes could be expected, as PEA3 expression is also strongly negatively linked to ER
expression (Table III). Finally, only NRG1 and MMP2 were associated to PEA3 gene independently to proliferation and ER
expression status. The positive correlations between the expression of PEA3 and these two genes, in a proliferation- and ER
-independent fashion, was confirmed on a larger series of 54 breast tumors, including tumors over-expressing, under-expressing and normally expressing PEA3 (data not shown).
This same series of 130 breast tumors had been tested previously for mRNA expression of various other genes known to be altered in breast cancer, i.e. BRCA2, MYC, CCND1, STMN1 (Stathmin), TERT, RB1, CGB, CGA, PS2, AR, PGR, CAV1, CXCR4, CXCR12 and CDH1 (E-cadherin) genes. We found a significant positive link, in a proliferation- and ER- independent fashion, between PEA3 and only the CGB gene, which encodes the human chorionic gonadotropin ß subunit (r = +0.662, P = 0.0008) (Table IV).
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Discussion |
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Variations are therefore observed in terms of frequency of PEA3 over-expression between our study and the two clinical studies published previously (25,26). It is noteworthy that our study examined PEA3 mRNA expression by real-time quantitative RTPCR analysis (14% of PEA3 over-expression; >3- fold representing over-expressing) whereas Benz et al. (25) examined PEA3 mRNA expression by in situ hybridation (76% of PEA3 over-expression; >2-fold) and Kinoshita et al. (26) examined PEA3 protein status (42% of PEA3 immunoreactivity-positive). In situ hybridation and immunohistochemistry assess specimens on an individual cell basis but have relatively little inter-laboratory standardization and are not accurate enough to quantify the full range of gene alterations. In consequence, further studies, using the same technical conditions, are necessary to determine the accurate frequency of PEA3 alteration in breast cancer.
In our study, significant links were observed between high PEA3 mRNA levels and SBR histopathological grade III, high MKI67 and ERBB2 mRNA levels and low ER mRNA levels. High PEA3 mRNA levels were not associated with a poor prognosis, suggesting that PEA3 is a marker of tumor aggressiveness rather than a prognostic factor in human breast cancer. Formally, these observations are consistent with a model in which PEA3 exerts a positive effect on proliferation of breast tumor cell. Such a model is difficult to reconcile with the genetic evidence that PEA3 is a tumor-suppressor gene whose over-expression should inhibit breast tumorigenesis in repressing ERBB2 gene expression (12). In this regard, our data are in agreement with those reported by Benz et al. (25), which also showed a coordinately up-regulation between PEA3 and ERBB2 in breast cancer, suggesting that PEA3 has rather a role of an oncogene. Taken together, these findings coupled with previous observations demonstrating increased PEA3 transcript levels in mouse metastatic mammary adenocarcinomas (22) as well as in mammary tumors of MMTV-neu transgenic mice (23) suggest that the use of PEA3 as a therapeutic agent may not be appropriate in breast cancer. Our data, in agreement with those of Shepherd et al. (23) rather support the use of therapeutic agents that inhibit the expression or activity of PEA3 in the treatment of breast cancer.
With respect to PEA3 expression in cell lines, our results are in total agreement with those reported recently by Baert et al. (21), who analyzed the PEA3 expression pattern in a large series of human breast cell lines by means of northern blotting. For example, the highest PEA3 expression levels observed by Baert and ourselves were in cell lines BT-20 and MDA-231.
We also studied the possible relationship between the expression of PEA3 gene and that of various candidate genes involved in the PEA3 pathway dysregulations observed in breast cancer. Among the 30 candidate genes tested, only the genes NRG1 and MMP2 were associated to PEA3 gene independently of proliferation and ER expression status. It is noteworthy that the positive links observed between PEA3 expression and two well known genes transactivated by PEA3 transcription factor, i.e. MMP2 and NRG1, suggests a link between PEA3 mRNA level and PEA3 protein activity. We also identified an additional gene (CGB gene) linked to PEA3 among 16 genes known to be dysregulated in breast tumors, but not known to be transactivated by the PEA3 transcription factor.
Several results of this study do not confirm those reported in the literature or relationships hypothesized by ourselves. (i) We did not find any association in our breast tumor series between PEA3 gene and the genes UPA, GJB2 (coding for Connexin 26) or TIMP1, which reveal several PEA3 sites localized within their promoters (6,8,11). (ii) We did not observe a link between the expressions of PEA3 and HGF (hepatocyte growth factor) as observed in vitro in oral squamous cell carcinoma cell (16). (iii) Our data did not suggest that PEA3 gene is epigenetically regulated by DNA methylation as observed in the in vitro model with conditional DNMT1-deficient primary mouse fibroblasts (18). Indeed, we did not observe links (independent of proliferation) between PEA3 and neither DNA methyltransferase genes (DNMT1, 3A and 3B) nor GRO1 and JUNB, which are highly co-up-regulated with PEA3 in DNMT1-deficient mouse cells.
Our results identified type I gelatinase (MMP2) as the major matrix metalloproteinase activated by PEA3 in breast tumors. Higashino et al. (9) showed previously, in transient expression assays, that PEA3 can activate MMP2 gene promoter. Expression of the chloramphenicol acetyltransferase reporter gene under the control of MMP2 promoter was increased >10-fold by co-transfection with a PEA3 expression vector.
Our results also identified NRG1 (heregulin) among six ErbB-specific ligands as a major growth factor, which up-regulates PEA3. Our in vivo study confirms the recently reported activation of PEA3 transcription by heregulin in an ERBB2-over-expressing breast cell line, i.e. SKBR3 cell line (15).
Finally, our results suggest the existence of an additional target gene of PEA3 transcription factor, which could be associated with breast tumorigenesis. Indeed, among 16 genes known to be dysregulated in breast tumors, we identified the CGB gene encoding the glycoprotein hormone ß subunit as a major target gene of PEA3 transcription factor. We have shown previously that CGB over-expression is associated with malignant breast transformation (33). It is noteworthy that Johnson and Jameson (34) have recently identified Ets2, another member of Ets-related transcription factors, as a new factor involved in cyclic AMP induction of the human CGB promoter in the JEG-3 placental cell line. It is possible that in breast tumors, PEA3 could bind to the same identified Ets-binding site that the Ets2 transcription factor binds to in the human CGB promoter.
Further studies (including cDNA microarray analysis) are necessary to identify the complete list of the PEA3-stimulated genes. In this regard, Howe et al. (35) recently suggested that the PEA3 factor might contribute to the up-regulation of COX-2 expression.
In conclusion, this study confirms the role of the PEA3 gene in breast tumorigenesis, and suggests that PEA3 is a marker of tumor aggressiveness rather than a prognostic factor in human breast cancer. Our results do not confirm that PEA3 is a tumor-suppressor gene that can act therapeutically in ERBB2 over-expressed tumors. Our results confirm the MMP2 gene and identify the NRG1 gene in the PEA3 pathway dysregulation observed in breast cancer. Finally, we hypothesize the existence of numerous other genes (such as CGB) transactivated by the PEA3 transcription factor in breast cancer.
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Notes |
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Acknowledgments |
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References |
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