Annexes
D.1 Article Annexe
From gene to oncogenesis,
the example of Ets transcription factors from the PEA3 group
Sébastien MAUEN1, Jean-Luc BAERT2, and Yvan de LAUNOIT*,1,2
1 Laboratoire de Virologie Moléculaire, Faculté de Médecine, Université Libre de Bruxelles, CP 614, 808 route de Lennik, 1070 Brussels, Belgium.
2 UMR 8117, CNRS / Université de Lille I / Institut Pasteur de Lille, Institut de Biologie de Lille, BP 447, 1 rue Calmette, 59021 Lille Cedex, France.
* To whom correspondence should be addressed:
Phone : +322 555 62 45 ; Fax : +322 555 62 57 ; E-mail : ylaunoit@ulb.ac.be
ABSTRACT
The PEA3 group of Ets transcription factors is composed of three highly conserved members : Erm, Er81 and Pea3. They regulate transcription of their target genes following post-translational modifications such as phosphorylation, acetylation, sumoylation and ubiquitinylation. Among their target genes are several matrix metalloproteases (MMP) which are enzymes degrading extracellular matrix during normal remodelling events and cancerous metastasis process. In fact, PEA3 group members are often overexpressed in different types of cancers which also overexpress these MMP and present a disseminating phenotype. This suggests that they play a key role in metastasis.
The ets genes are found in the most of known multi-cellular organisms (Dittmer and Nordheim, 1998; Sharrocks et al., 1997). They encode a family of transcription factors including more than 30 members, which are, in the most cases, transcriptional activators, and in some rare exceptions, transcriptional repressors (Graves and Petersen, 1998). These transcription factors share a conserved motif of 85 amino acids, the ETS domain, which is the signature of the family (Graves and Petersen, 1998). This domain permits the transcription factors to bind DNA on a sequence of 9 nucleotides with the central consensus core 5’- GGAA/T-3’: the “ETS Binding Site” (EBS) (Sharrocks et al., 1997). The sequences flanking this central motif determine the binding specificity for each of the Ets family members (Oikawa and Yamada, 2003; Sharrocks, 2001). Phylogenetically, this transcription factor family is classified in 13 groups according to sequence alignment of the ETS domain, the position of this domain in the protein and the presence of other specific conserved functional domains (Laudet et al., 1999).
The PEA3 group
The PEA3 group is composed of three members: Erm (also called Etv5) (Chotteau- Lelievre et al., 1997; Monte et al., 1994; Nakae et al., 1995), Er81 (also called Etv1) (Brown and McKnight, 1992; Jeon et al., 1995; Monte et al., 1995) and Pea3 (also called E1Af or Etv4) (Friedman et al., 1994; Higashino et al., 1993; Xin et al., 1992), which share in common the highly conserved ETS domain and two conserved transactivating domains located at the amino- (AD) and the carboxy-terminal (Ct) termini (de Launoit et al., 1997).
Human and mouse er81, erm and pea3 genes are structured in 13-14 exons distributed on more than 15 kbp of genomic DNA (Coutte et al., 1999a; Coutte et al., 1999b; Monte et al., 1996). In human, erm is localised on chromosome 3 at position 3q27-29 (Monte et al., 1994;
Nakae et al., 1995), e1af at position 17q21 and er81 at position 7q21 (Jeon et al., 1995).
Only partial structural studies are currently available on the Ets family proteins as well as on the PEA3 group members. The crystal structure of the ETS domain was elucidated, with or without binding DNA, and it consists of a winged helix-turn-helix motif (Sharrocks et al., 1997). The Erm AD transactivating domain was also studied by circular dichroïsm revealing that 15 amino acids of this highly acidic domain were structured in a α-helix (Defossez et al., 1997). We recently performed a global structural analysis of Erm by combining circular dichroïsm and infrared spectrometry methods. Comparison of these data with current structural prediction algorithms confirms the highly structured ETS domain and suggests that each of the transactivating domains also contains a α-helix. In contrast, the 250 residue central domain seems to be very weakly structured (Mauen et al., 2006).
In most of the cases, the PEA3 group members are described as transcriptional activators (Laget et al., 1996; Monte et al., 1995; Xin et al., 1992) with two transcriptional domains (AD and Ct), which are able to synergize. Negative regulatory domains encompass the transactivating domains in Pea3 and Erm (Bojovic and Hassell, 2001; Laget et al., 1996).
Recent data on Erm and Pea3 suggest that the Ct domain also plays a role in the stability of the protein (Baert et al., 2006; Takahashi et al., 2005).
As for the other Ets transcription factors, it would be unlikely that these transcription factors alone regulate transcription of their target genes. They in fact interact with transcriptional partners. Thus Erm associates with the basal transcription complex proteins TAFII60, TBP and TAFII40 (Defossez et al., 1997). Erm also interacts with the androgen receptor, which leads to the repression of Ets-mediated transactivation (Schneikert et al., 1996). One of the AP1 complex proteins, c-Jun, also physically interacts with the Ct domain of Erm to synergistically enhance transcriptional activation (Nakae et al., 1995). Furthermore, Er81 (Goel and Janknecht, 2003), Pea3 (Liu et al., 2004) and Erm (unpublished) interact with the p300 transcriptional co-activator, an enzyme with histone acetyltransferase activity (HAT). Er81 also interacts with the p300 associated partner P/CAF (Goel and Janknecht, 2003). More recently, the SUMO conjugating enzyme Ubc9 was also shown to interact with Er81 (Gocke et al., 2005) and Erm (Degerny et al., 2005). Interestingly, Pea3 has recently been shown to interact with the USF-1 transcription factor to synergistically regulate expression of the bax gene without directly binding DNA (Firlej et al., 2005).
Schematic representation of a PEA3 group prototype with its different functional domains and regulating pathways. The ETS-domain is responsible for DNA-binding. The two TAD correspond to the transcriptional activation domains. Post-translational modifications up- (upper) or down- (lower) regulating the transcriptional activity of the PEA3 group members on their target genes. Ac : acetyl group ; P : phosphate group, SUMO : sumo peptide; Ub : ubiquitin.
PEA3-dependent transcriptional activity is regulated by post-translational modifications As many transcription factors, the PEA3 group members are post-translationaly modified in order to regulate the transactivation capacities. The most common modifications retrieved in the PEA3 group proteins is phosphorylation, as they are target of the MAPK pathway including Ras, Raf-1, MEK and the MAPK ERK-1 and ERK-2. Phosphorylation of specific serine and threonine residues generally induces increased transactivation capacities of the PEA3 group member (Bosc et al., 2001; Janknecht, 1996; Janknecht et al., 1996; O'Hagan et al., 1996). Moreover, Erm and Er81 are also phosphorylated through the PKA-mediated pathway (Baert et al., 2002; Brown et al., 1998; Coutte et al., 1999b; Janknecht et al., 1996).
This post-translational modification which occurs at the edge of the ETS domain, induces both activation of the transcriptional capacity and loss of DNA binding (Baert et al., 2002;
TAD ETS TAD
CBP/p300
K K
PKA MAPK
P P
S T
P T P
S P
MSK 1
S S
?
K K K K
K P
SUMO SUMO SUMO SUMO
Ac Ac
Ub
Wu and Janknecht, 2002). This is a very rare example of increase of the transactivation capacity of a transcription factor with a dramatic decrease of its capacity to bind DNA.
Post-translational modifications to lysines also play crucial roles in the regulation of transcription, generally on histone proteins but also on transcription factors. This is the case for the PEA3 group members for which activation of p300 through the Ras/MAPK pathway results in Er81 acetylation; this latter leading to an increase of DNA binding as well as transcriptional activity of this factor (Goel and Janknecht, 2003). Recently, Er81 and Erm have been shown to be submitted to another lysine modification; i.e. SUMO modification via the Ubc9 SUMO conjugating enzyme (Degerny et al., 2005; Gocke et al., 2005). More precisely, Erm contains four different functional sites, on which the polypeptide SUMO can be covalently linked. This modification leads to an inhibition of the Erm-dependent transactivation without affecting the protein localisation as well as the DNA-binding ability (Degerny et al., 2005). The SUMO modification mechanism is very closely related to the ubiquitination modification mechanism, which also consists in the addition of one or more ubiquitins to targeted lysines. In the case of the PEA3 group, Erm and Pea3 are conjugated to ubiquitin and degraded via the ubiquitin-proteasome pathway which probably regulates the activity of these transcription factors (Baert et al., 2006 ; Takahashi et al., 2005).
Functional roles during embryogenesis
These transcription factors are expressed in multiple mouse developing organs with the three germ layer origin, such as lung, salivary glands, kidney, gut… However, when co- expressed in the same organ, they are generally present in different tissues. For example, pea3 and erm are preferentially and primarily expressed in developing epithelial cells of the salivary gland or lung, whereas er81 expression is rather restricted to the surrounding mesenchymal compartment (Chotteau-Lelievre et al., 1997; Chotteau-Lelievre et al., 2001).
The general expression pattern for these three genes during branching morphogenesis is the following : erm is expressed in the distal portion of epithelial buds, pea3 in a more restricted region of the growing epithelium and, er81 in the adjacent mesenchyme. This pattern is not restricted to branching morphogenesis but is found during conversion of mesenchyme to epithelium (e.g in kidney). This phenomenon is generally associated to a dramatic decrease of both erm and pea3 expression, and a dominant-negative form of Erm induces severe abnormalities in mouse lung development due to an inhibition or a delay of distal precursor cell type differentiation (Liu et al., 2003).
During embryogenesis, expression of the PEA3 group members is not restricted to glandular organs, since er81 and pea3 are both expressed in the nervous system. pea3 and er81 are differentially expressed in the chick spinal cord, thus allowing to define specific motor neuron pools and subset of muscle sensory afferent neurons. In fact, motor and sensory neurons innerving the same muscle express the same PEA3 group member, indicating the requirement of a certain expression pattern for functional circuits’ formation (Arber et al., 2000; Lin et al., 1998). Actually, Pea3, downstream GDNF induction, is required for correct arborisation of motor neuron pool; in pea3 or GDNF mutant, some specific muscles show their innervations dramatically reduced (Haase et al., 2002; Livet et al., 2002). Null er81 mice also fail to establish terminal branching of proprioreceptive sensory neurons into the ventral spinal cord (Arber et al., 2000). Altogether, these data suggest that expression of the PEA3 group members is involved in multiple events during embryogenesis like epithelial- mesenchymal interactions inducing tissue remodelling events, cell proliferation and also cell differentiation.
In mouse adult tissues, erm and, in a lesser extent, er81 are almost ubiquitously expressed at a high level in brain, colon, testis and lung for erm and brain, lung and heart for er81 (Chotteau- Lelievre et al., 1997; Monte et al., 1994). Mouse pea3 mRNA expression is more restricted than for the two other members, with a main expression pattern in brain (Chotteau-Lelievre et al., 1997) as well as in epididymis (Xin et al., 1992). These expression profiles have been confirmed in human tissues (Monte et al., 1994; Monte et al., 1995). Null erm male mice present a phenotype of spermatogenesis disruption leading to sterility, mainly due to the fact that Erm protein is required for spermatogonial stem cell self-renewal (Chen et al., 2005).
Null pea3 male mice also present a severe male sexual dysfunction with ejaculatory dysfunction probably due to neuronal defect in the penis (Laing et al., 2000).
Cancer : link with the metastasis process
As described below, these factors are expressed during remodelling events, cell differentiation and proliferation. It is thus of interest to know which genes they target. Many studies have reported their implication in the transcriptional regulation of matrix metalloprotease enzymes (MMP). These latter are involved in the degradation of the extracellular matrix to permit tissue rearrangement, a common phenomenon in cancer metastasis. In reporter assay experiments, the PEA3 group members enhance transcription of different MMP promoters, such as the human stromelysin-1, the type I and IV collagenases (Higashino et al., 1995) and Matrilysin (Crawford et al., 2001). In vivo, ectopic expression of
Er81 in the normal mouse mammary gland induces up-regulation of the urokinase plasminogen activator gene (Netzer et al., 2002). They also can positively regulate the promoters of vimentin (Chen et al., 1996), the intercellular adhesion molecule ICAM-1 (de Launoit et al., 1998), osteopontin (El-Tanani et al., 2004) and cyclooxygenase-2 (Howe et al., 2001) genes. This ability to enhance such promoters lets suggest that they could play a role in tumourigenesis. In fact, PEA3-overexpressing cell lines present a high metastasis potential correlated with high expression of several MMPs (Habelhah et al., 1999; Hida et al., 1997b;
Kaya et al., 1996; Shindoh et al., 1996), whereas pea3 knock-down reduces MMP-1 and 9 expression (Hida et al., 1997a). Here below, we will summarize the current state-of-the-art of the roles of the PEA3 group members in cancers of different origins.
In the oral tract, pea3 is highly expressed in squamous carcinoma cell lines which are of high invasive phenotype and express high levels of type I and IV collagenases (respectively MMP- 1 and MMP-9) (Shindoh et al., 1996). Moreover, transfected cells with pea3 antisense exhibit a decrease of the expression of the two MMPs mentioned above (Hida et al., 1997a). In these cells, Pea3-induced MMP expression is also stimulated by the hepatocyte growth factor (HGF) (Hanzawa et al., 2000). In in vivo tumours, pea3 mRNA is detected by in situ hybridisation in the majority of the invasive squamous cell carcinoma specimens studied, and increased expression of Pea3 is correlated with nodal metastasis (Hida et al., 1997b). It has also been recently demonstrated in these cells that Pea3 is able to upregulate MT1-MMP expression, a trans-membrane MMP, which activates the proMMP-2, and thus indirectly activates MMP-2 (Izumiyama et al., 2005).
In lung, in contrast to pea3, both erm and er81 are highly expressed in normal adult lung (Chotteau-Lelievre et al., 1997; Monte et al., 1994; Monte et al., 1995). Interestingly, Pea3 is currently the only member of the PEA3 group which is involved in the lung cancer process. In non-small-cell lung cancers (NSCLCs) which are characterized by cell invasiveness, pea3 mRNA is expressed in the primary tumours and not in the surrounding normal tissues. Ectopic expression of pea3 in NSCLC cells significantly increases in vitro cell motility and invasion (Hiroumi et al., 2001).
Previous studies described the involvement of β1,4-galactosyltransferase I (GalT I) in cellular processes such as cell migration (Appeddu and Shur, 1994). If little is known on the role of GalT I in metastasis, several studies have shown that its increased expression at the cell surface is specific of highly metastasis cell lines (Passaniti and Hart, 1990; Penno et al.,
1989). Recently, Pea3 and GalT I expression has been correlated to a high metastasis phenotype in lung cancer cells (Zhu et al., 2005).
In the gastrointestinal tract, the PEA3 group members are also expressed. In the case of gastric cancer, pea3 mRNA expression is significantly correlated to tumour invasiveness and recurrence, indicating a bad prognosis for patient survival (Yamamoto et al., 2004).
Moreover, in this study, MMP-7, a marker of invasiveness in gastric cancer (Liu et al., 2002), and pea3 mRNA expression correlates in cancer tissues. Moreover, they co-localize at the
“invasive front” and, when pea3 expression is knocked-down, matrilysin expression is dramatically decreased, and cells are less invasive in vitro.
In human hepatocellular carcinoma (HCC), the interleukin IL-8, which is a potent angiogenic factor, is constitutively induced by Pea3 in association with the transcription factor AP-1. Moreover, pea3 and IL-8 are both expressed in HCC, and not in the surrounding tissues, thus indicating a potential role of Pea3 in IL-8-induced hepatoma tumorigenesis (Iguchi et al., 2000).
Promoters of several MMP involved in human colorectal adenocarcinomas possess specific DNA binding sites for the PEA3 group members. This is the case for the meprin beta (Matters and Bond, 1999) as well as the matrilysin (MMP-7) (For a review see ref.(Wilson and Matrisian, 1996); for this latter Pea3 co-operate with AP1. The β-catenin gene is also regulated by the PEA3 group members in mouse colon tumour cells, since both pea3 and erm are co-expressed in these cells and they both, in synergy with β-catenin, upregulate the MMP- 7 promoter; thus finally contributing to tumorigenesis (Crawford et al., 2001). In colorectal tumours, pea3 expression is also significantly correlated with invasion, metastasis and advanced tumour stages, and co-expressed with β-catenin, MMP-1 and MMP-7; these two latter genes being down-regulated by pea3 knock-down (Horiuchi et al., 2003). Moreover, recent immunochemistry studies also demonstrate that pea3 expression, in premalignant tissues as colorectal adenomas, is correlated to over-expression of, not only MMPs, but also COX-2 and iNos, two factors involved in tumour angiogenesis (Boedefeld et al., 2005; Liu et al., 2004; Nosho et al., 2005). Altogether, these data suggest that Pea3, in association with β- catenin, plays a role in colorectal tumour progression, by targeting specific key target genes.
In gynaecologic tissues, these factors play important roles. er81 is weakly expressed in human ovary whereas erm is not. However erm expression is found in human ovarian adenocarcinoma (Monte et al., 1994; Monte et al., 1995). In the mouse ovary, only erm is expressed at low level (Chotteau-Lelievre et al., 1997). In ovarian and endometrial cancer cell lines, Er81 activates transcription of Smad7, which can participate to inhibition of the TGF-β signalling pathway with anti-proliferative effects (Dowdy et al., 2003). Pea3 is also implicated in ovarian carcinoma, since expressed in the majority of the carcinomas studied.
Interestingly, intense pea3 expression in the stroma is also detected in the vicinity of grade II and III tumours and is correlated to MMP-2 expression within the carcinoma cells, thus illustrating the dialogue between these two compartments. Expression of pea3 is also correlated to MMP-1, EMMPRIN (a MMP inducer), angiogenic factors such as bFGF, IL-8 and integrins (Davidson et al., 2003; Davidson et al., 2004).
The role of the PEA3 group members in normal and pathological mammary development has been extensively studied these last years. They are indeed expressed at different steps of the mouse mammary gland development. During embryogenesis, they are highly expressed in the epithelial buds of the mammary gland (Chotteau-Lelievre et al., 1997), and after birth, pea3 and erm mRNAs are detected throughout all stages of mammary gland development, whereas er81 is only present in pubescent gland. During puberty, pea3 and erm expression is correlated to ductal elongation and branching (Chotteau-Lelievre et al., 2003). Supporting this observation, normal murine mammary cells over-expressing the PEA3 group members show an increased capacity to form in vitro branching duct-like structures (Chotteau-Lelievre et al., 2003).
We described here above that PEA3 group expression in cancers from the respiratory and gastrointestinal tracts is highly correlated to the bad prognosis of the patient. In mammary cancer, expression of the HER2/Neu growth factor receptor, whose expression is correlated to bad prognosis, has open the suggestion of its putative co-expression with the transcription factors from the PEA3 group. This relationship has been widely studied. The first experimental evidence was the over-expression of pea3 in neu-induced mouse mammary primary adenocarcinomas, as well as the corresponding metastatic lesions (Trimble et al., 1993). er81 and erm are also expressed in such tumours (Galang et al., 2004). In contrast, inhibition of the transcriptional activity of the PEA3 group members by expressing a dominant negative form in these transgenic mice delays the tumourigenesis onset and reduces tumour progression (Shepherd et al., 2001). In human breast cell lines, the three factors are highly expressed, more particularly in estradiol receptor negative cells, which display an
aggressive phenotype in nude mice (Baert et al., 1997). Although over-expression of HER2/neu is only observed in 20-30% of all breast carcinomas (Singleton and Strickler, 1992), pea3 expression is observed in more than 90% of HER2/neu positive breast carcinomas, and less than 50% of the HER2/neu negative ones. Moreover, pea3 and neu expression is positively correlated with invasive breast tumours (Benz et al., 1997; Bieche et al., 2004). However, provocative data concerning pea3 expression in human breast carcinomas shows better overall survival when this factor is present (Kinoshita et al., 2002).
The molecular mechanism by which Pea3 regulates the HER2/neu gene is at the transcriptional level, since binding to the promoter (Scott et al., 1994) and enhancing a reporter plasmid containing this promoter (Benz et al., 1997). Moreover, HER2/neu is able to enhance Pea3 transactivation through the ERK and SAPK/JNK pathways (Benz et al., 1997;
O'Hagan et al., 1996) and thus can lead to its own transactivation. Pea3 also enhances transcription of other genes involved in mammary metastasis; i.e. the osteopontin in rat mammary cell lines (El-Tanani et al., 2004), and the Muc4/sialomucin complex in HC11 normal mouse mammary cells (Perez et al., 2003), cyclooxygenase-2 (COX-2) in human breast epithelial cell lines 184B5 and 184B5/HER (Subbaramaiah et al., 2002). HER2/neu- induced activation also targets Er81 through the Ras-Raf-MAPK pathway, thus increasing the transcriptional activity of this transcription factor on different target genes such as MMP-1, the TGFbeta inhibitor Smad7, and the telomerase reverse transcriptase hTERT (Bosc et al., 2001; Dowdy et al., 2003; Goueli and Janknecht, 2004) Whatever it is, two provocative studies from the same group reported that Pea3 can act on the HER2/neu promoter as a transcriptional repressor in specific mammary cancer cell lines (Hung and Wang, 2000; Xing et al., 2000). Finally, recent studies have demonstrated that erm expression is correlated to invasiveness and poor prognosis in breast cancer (Chotteau-Lelievre et al., 2004) and to invasiveness in endometrial carcinoma (Planaguma et al., 2005). The functional role of this transcription factor in these cancers should be defined in more details.
The PEA3 group members also involved in chromosomal rearrangement in cancer
To be exhaustive on the roles of the PEA3 group members in human cancer, we should mention here that in very few cases, genes of these transcription factors are rearranged.
In fact, Ewing’s sarcoma, which is an aggressive neoplasia affecting predominantly children and young adults, results from a chromosomal translocation between an ets gene and the ews RNA-binding protein gene. Two members of the ets gene family are currently described to be fused with ews : fli-1 in 85% cases (Delattre et al., 1992) and erg in 14% cases (Sorensen et
al., 1994). However, er81 (Jeon et al., 1995), pea3 (Kaneko et al., 1996; Urano et al., 1996) and fev (Peter et al., 1997) have also been characterized by the formation of a fusion protein containing the amino–terminal transactivation domain of EWS and the ETS DNA-binding domain from the ets gene (For a review see ref.(Arvand and Denny, 2001). These chimeric Ews-Ets proteins display an increased potential to transactivate the promoters of Ets target genes when compared to the wild-type Ets proteins (Bailly et al., 1994). Moreover, due to the ews promoter, these chimeras also possess a ubiquitous acute expression, thus suggesting that in Ewing’s sarcoma, Ets-target genes could be up-regulated by the high amount of chimera.
However, although Ews-Er81 is able to in vitro transactivate the MMP-1 promoter in association with p300 and c-Jun (Fuchs et al., 2003), opposite conclusions are drawn in vivo.
In fact, Ewing’s sarcoma expressing ews-pea3 doesn’t express MMP-1 and MMP-3, probably because of a loss of accessibility to EBS for the chimera (Yabe et al., 2002). Both Ews-Er81 and Ews-Pea3 can also increase the transcription of hTERT, a catalytic subunit of telomerase which induces telomerase activities and thus cell immortalization (Fuchs et al., 2004; Shindoh et al., 2004).
Finally, er81 (etv1), as well as erg, were identified as rearranged in prostate cancers with gene fusion of the 5’ untranslated region of the TMPRSS2 gene to er81 or erg with outlier expression. In fact, 23 of the 29 prostate cancer samples studied harbour rearrangements in erg or er81 and the androgen-responsive promoter elements of TMPRSS2 mediate the overexpression of Ets family members in these cells. These very recent data have implications in the molecular diagnosis and treatment of prostate cancer (Tomlins et al., 2005).
In conclusion, the PEA3 group members are functional transcription factors of the Ets family, which play a role in the human cancer. More particularly, they regulate the expression of target genes involved in the metastasis process.
ACKNOWLEDGEMENTS
This work was carried out thanks to grants awarded by the "Centre National de la Recherche Scientifique" (France), the “Institut Pasteur de Lille”, the "Association pour la Recherche contre le Cancer" (France), the “Ligue Nationale Contre le Cancer” (Comité Nord, France), « the Conseil Régional Nord/Pas-de-Calais » (France, the European Regional Developmental Fund, the “Fonds National de la Recherche Scientifique” (FNRS, Belgium) and the “Action de Recherche Concertée (Communauté Française de Belgique)” (Belgium).
