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Human Molecular Genetics Advance Access originally published online on March 6, 2007
Human Molecular Genetics 2007 16(9):1058-1071; doi:10.1093/hmg/ddm053
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Comparative genomic and functional analyses reveal a novel cis-acting PTEN regulatory element as a highly conserved functional E-box motif deleted in Cowden syndrome

Marcus G. Pezzolesi1,2, Kevin M. Zbuk1,2, Kristin A. Waite1,2,3 and Charis Eng1,2,3,4,5,*

1 Genomic Medicine Institute, 2 Lerner Research Institute and 3 Taussig Cancer Center, Cleveland Clinic Foundation, 9500 Euclid Avenue, NE-50, Cleveland, OH 44195, USA, 4 Department of Genetics and 5 CASE Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA

* To whom correspondence should be addressed. Tel: +1 2164443440; Fax: +1 2166360655; Email: engc{at}ccf.org

Received February 4, 2007; Accepted February 28, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Germline mutations in PTEN, encoding a phosphatase on 10q23, cause Cowden syndrome (CS) and Bannayan–Riley–Ruvalcaba syndrome (BRRS). Approximately, 10% of CS-related PTEN mutations occur in the PTEN promoter and 11% of BRRS-related mutations include large deletions, often favoring the gene's 5' end (exon 1, promoter). In order to better understand the mechanism(s) underlying the deregulation of PTEN in these syndromes, it is important that functional cis-regulatory elements be identified. We employed a comparative genomic approach combined with molecular genetic techniques to identify a highly conserved sequence upstream of the PTEN promoter, sharing 80% sequence identity among Homo sapiens, Mus musculus and Rattus norvegicus. Within this region, we identified a canonical E-box sequence (CACGTG) located at position –2181 to –2176, approximately 800 bp upstream of the PTEN core promoter and more than 1.1 kb upstream of its minimal promoter region (located at –958 to –821). In vitro assays suggest that this motif is recognized by members of the basic region-helix-loop-helix-leucine-zipper (bHLH-LZ) transcription factor family, USF1 and USF2, and reporter assays indicate that this novel E-box is involved in mediating PTEN transcriptional activation. Four of 30 CS/CS-like patients, without previously identified PTEN mutations, were found with germline deletions of the E-box element. Of the four, three had deletions stretching to exon 1, but not 3' of it; importantly, one classic CS patient harbored a germline deletion localizing to this E-box region, further affirming the role of this element in PTEN's regulation and deregulation, and its contribution to the pathogenesis of CS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Germline mutations in the 10q23-located gene encoding phosphatase and tensin homolog deleted on chromosome 10 [PTEN (MIM 601728 [OMIM] )] have been found in 85% of probands with Cowden syndrome [CS (MIM 158350 [OMIM] )] and 65% of probands with Bannayan–Riley–Ruvalcaba syndrome [BRRS (MIM153480)] (1–3). PTEN, a ubiquitously expressed tumor suppressor dual-specificity phosphatase antagonizes the phosphatidylinositol-triphosphate kinase (PI3K) signaling pathway through its lipid phosphatase activity, resulting in the subsequent inhibition of the Akt proto-oncogene (4–7). PTEN's protein phosphatase activity negatively regulates the mitogen-activated protein kinase (MAPK) pathway (8,9). Inactivation or loss of PTEN function results in increased cell survival and uncontrolled cellular proliferation mediated by these pathways and, as is seen in many human cancers, ultimately resulting in neoplasia.

PTEN is believed to be a constitutively active protein, whose sufficient activity is dependent upon protein levels (10). Despite this fact, and PTEN's significant roles in regulating the cell cycle and in the malignant transformation associated with carcinogenesis, relatively little is known about the mechanisms that govern transcriptional regulation of PTEN expression, and virtually nothing is known about its transcriptional regulation in human heritable disorders such as CS and BRRS. Previous in vitro studies have identified functional consensus binding sites for transcriptional activators p53 and early growth response-1 (EGR1) in the PTEN core promoter region [nucleotide (nt) position –1344 to –745] (11,12). Additional transcription factors have been shown to be involved in regulating PTEN transcription, including peroxisome proliferator-activated receptor gamma (PPAR{gamma}), nuclear factor kappa B (NF{kappa}B), c-Jun and, most recently, CBF-1 (13–16). However, for most of these, the precise mechanisms of transcriptional regulation remain unclear.

Together with the data from the Human Genome Project, sequence information from several non-human vertebrate genomes is being used to identify novel regulatory elements in previously uncharacterized, non-coding DNA (17–19). Comparative sequence analysis approaches have identified highly conserved regions that contain functionally important elements involved in the regulation of several human genes, including IL-4, IL-13, IL-5, SCL, IFN-{gamma} and BRCA1 (20–23). Using a similar approach, here, we set out to identify novel functional cis-acting regulatory elements along and around the PTEN locus, an exercise which is directly germane to the observation that germline mutations in the PTEN promoter occur in 10% of mutation positive CS, and large deletions, favoring the 5' end of PTEN (exon 1) and upstream of the gene (i.e. the promoter region), occur in 11% of BRRS patients (3).

In using a comparative genomic approach, we identified a highly conserved sequence, sharing 80% sequence identity, among the Homo sapiens, Mus musculus and Rattus norvegicus PTEN locus. Within this region, we identified a canonical E-box sequence (CACGTG) located at position –2181 to –2176, approximately 800 bp upstream of the PTEN core promoter and more than 1.1 kb upstream of its minimal promoter region (located at –958 to –821). In vitro assays suggest that this motif is recognized and bound by members of the basic region-helix-loop-helix-leucine-zipper (bHLH-LZ) transcription factor family, USF1 and USF2, and is involved in the transcriptional activation of PTEN. Furthermore, we identified one CS patient with a hemizygous germline deletion which localizes exclusively to this highly conserved region upstream of PTEN.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of novel DNA–protein interactions at a highly conserved E-box element upstream of the PTEN promoter
Multi-species comparative genomic analysis was used to identify highly conserved regions across a 163 kb region spanning the entire PTEN gene and including 30 kb of flanking sequence in three distantly related species: Homo sapiens, Mus musculus and Rattus norvegicus. This analysis yielded several highly conserved regions (>70% identity, across 100 bp), with the majority (20/25) localized to a region upstream of the PTEN promoter and within the gene's first intron (Fig. 1A). Of the regions displaying sequence homology, one particular region, located at position –2250 to –2151, exhibited approximately 80% sequence identity across 102 bp of DNA among all three species (Fig. 1B), compared with 70–77% for the other regions. Because of this extensive conservation, we chose to examine this region further.


Figure 1
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  		Figure 1. Comparative genomic analysis reveals a highly conserved E-box upstream of the PTEN locus. (A) Schematic diagram of the PTEN gene and the mVISTA alignment from our three-species comparative genomic analysis. A 24 kb region, including 3 kb upstream of PTEN and approximately 20 kb of intron 1, is provided. The circled area indicates the region corresponding to –2262 to –2151. (B) Three-species nucleotide alignment of this region. The highly conserved E-box element (located at position –2181 to –2176) is underlined. Asterisks indicate conserved nucleotides.

 
To identify potential DNA–protein complexes formed at this conserved site, we performed mobility shift assays, utilizing HeLa nuclear extract and a PCR-amplified DNA probe which spans the conserved site, inclusive of nucleotides –2262 to –2151. We found that this region could indeed bind to nuclear protein, as a single retarded band, indicative of the formation of a DNA–protein complex, was observed (Fig. 2A). The formation of this complex was specifically inhibited in the presence of 100-molar excess of the unlabeled DNA probe, but not by an excess of a non-specific unlabeled DNA probe (Fig. 2A, lanes 3 and 4). In order to better localize the nucleotides involved in this complex, we performed a subsequent mobility shift assay using overlapping DNA probes which spans –2262 to –2195 (A) and –2218 to –2151 (B) (Fig. 2B). A complex is formed using the –2218 to –2151 probe, however, no DNA–protein complex formation is observed using the –2262 to –2195 probe (Fig. 2B, lanes 2 and 6). The DNA–protein complex formed with the –2218 to –2151 probe is specifically competed with an excess of the unlabeled DNA probe, but not by an excess of the non-specific unlabeled DNA probe (Fig. 2B, lanes 7 and 8). These data strongly suggest that the DNA–protein binding observed at this conserved site is localized to the nucleotides spanning position –2218 to –2151.


Figure 2
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  		Figure 2. Identification of a novel USF-specific DNA–protein interaction upstream of the PTEN promoter. (A) Electrophoretic mobility shift assay (EMSA) using a probe spanning the –2262 to –2151 region. Lane 1: radiolabeled probe only (–); lane 2: radiolabeled probe plus HeLa nuclear extract (+); lane 3: radiolabeled probe, HeLa nuclear extract and 100-molar excess of unlabeled specific competitor probe (Sp); lane 4: radiolabeled probe, HeLa nuclear extract and 100-molar excess of unlabeled non-specific (PHLPP exon 14) competitor probe (NS). (B) EMSA using probes spanning –2262 to –2195 (A) and –2218 to –2151 (B). Lanes 1 and 5, radiolabeled probe only (–); lanes 2 and 6, radiolabeled probe plus HeLa nuclear extract (+); lanes 3 and 7, radiolabeled probe, HeLa nuclear extract and 100-molar excess of unlabeled specific competitor probe (Sp); lanes 4 and 8, radiolabeled probe, HeLa nuclear extract and 100-molar excess of unlabeled non-specific (PHLPP exon 14, 274 bp amplicon) competitor probe (NS). (C) Binding observed at –2262 to –2151 was assessed using a series of specific and non-specific competitor probes. Lane 1, radiolabeled probe only (–); lane 2, radiolabeled probe plus HeLa nuclear extract (+). In lanes 3–8, radiolabeled probe, HeLa nuclear extract plus 100-molar excess of the following unlabeled competitors were added; lane 3: –2262 to –2151 [specific, 112 bp (Sp)], lane 4: –2200 to –2167 [specific, 34 bp (Sp)], lane 5: PHLPP exon 14 [non-specific, 274 bp (NS)], lane 6: PTEN exon 5 [non-specific, 25 bp (NS)], lane 7: Myc–Max consensus [specific, 26 bp (Con)], lane 8: Myc–Max mutant [non-specific, 26 bp (MT)]. (D) Binding at the putative E-box located at position –2181 to –2176 was evaluated using the –2262 to –2151 radiolabeled probe and mutated competitor probes. Lane 1: radiolabeled probe only (–), lane 2: radiolabeled probe plus HeLa nuclear extract (+). In lanes 3–9, radiolabeled probe, HeLa nuclear extract plus 100-molar excess of unlabeled specific competitor (Sp) and mutated competitor probes MT1 through MT6 were added, respectively.

 
Several putative transcription factor recognition sequences exist within the –2218 to –2151 region. Among these is a putative canonical E-box (CACGTG), located at position –2181 to –2176, to which several proteins are known to bind. To aid in identifying the most likely candidate proteins involved in the formation of this complex, we relied on the TESS transcription factor prediction software program (http://www.cbil.upenn.edu/cgi-bin/tess). Among the most significant predictions were members of the bHLH-LZ family of transcription factors; including upstream stimulatory factor (USF), Myc, Myc-associated factor X (Max) and transcription factor E3 (TFE-3). bHLH-LZ proteins, also known as E proteins, specifically bind to DNA sequences containing the E-box consensus sequence, which is minimally defined by the hexameric CANNTG motif (24).

To determine whether the binding we observed along this fragment was occurring at this E-box element, we performed a series of mobility shift assays using various non-specific cold competitor probes. For the initial experiments, we utilized a 34-nt specific oligonucleotide probe (spanning position –2200 to –2167) containing the E-box and commercially available Myc–Max consensus and mutant oligonucleotide probes, the former of which contains the consensus CACGTG E-box element, while the latter contains mutations of the two 3' nucleotides (CACGTG to CACGGA). In the presence of an excess of the –2200 to –2167 specific probe, formation of the DNA–protein complex was inhibited (Fig. 2C, lane 4). Futhermore, an excess of the Myc–Max consensus oligonucleotide also efficiently inhibited formation of the DNA–protein complex at this site, while an excess of the mutant oligonucleotide did not (Fig. 2C, lanes 7 and 8). These date suggest that the complex formed along this fragment occurs at the putative E-box located upstream of the PTEN promoter at position –2181 to –2176.

Identification of USF-specific binding at the –2181 to –2176 E-box element
It has previously been shown that the binding preferences of USF and Myc/Max to DNA are determined by the sequence flanking the core consensus E-box element (25–27). Using a specific competitor oligonucleotide probe, containing the core consensus E-box flanked by native sequence, and various mutant oligonucleotide competitor probes (Table 1), we chose to exploit this feature in order to discriminate which bHLH-LZ family members are likely involved in the formation of the DNA–protein complex observed at position –2181 to –2176. The mutant competitor probes contained mutated nucleotides either within the core E-box sequence or in the nucleotides flanking the consensus motif. Mutant oligonucleotides MT1, MT2 and MT3 contain mutated 5' half-sites (AattGTGA), 3' half-sites (ACACattA) or complete core (AattattA) sequences, respectively. MT4 (ACAgcTGA) contains an inversion of the core sequence's two central nucleotides, a nucleotide change tolerated by USF but not Myc/Max dimers (25). Both MT5 (tCACGTGA) and MT6 (cCtCACGTGACg) contain mutations in regions flanking the core sequence which disfavors the binding of Myc/Max and Max/Max complexes (28). An excess of either of the E-box half-site mutant oligonucleotides are able to diminish binding at this site (Fig. 2D, lanes 4 and 5), while MT3, with a completely mutated E-box motif, is unable to compete with the –2262 to –2151 radiolabeled probe. In contrast, the MT4, MT5 and MT6 mutant oligonucleotides, which favor USF binding, all efficiently inhibit formation of the DNA–protein complex, suggesting that the complex formed at the –2181 to –2176 E-box is due to the binding of USF.


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  		Table 1. Mutant oligonucleotide competitor probes/sequence of mutant reporter constructs

 
The –2181 to –2176 E-box element is involved in transcriptional activation
To determine the functional significance of the –2181 to –2176 E-box, we chose to examine the conserved region (position –2262 to –2151) using a luciferase reporter assay. The full-length PTEN promoter was subcloned into the pGL3.1-Basic vector (pGL3-B-FL). In addition, the –2262 to –2151 conserved region was subcloned upstream of the full-length PTEN promoter and into the pGL3-B-FL vector (pGL3-B-FL-2262-WT) (Fig. 3A). Both constructs, along with the pGL3-B empty vector, were transiently transfected into HeLa and MCF-7 cells and assayed for luciferase activity. The luciferase activity of the pGL3-B-FL and pGL3-B-FL-2262-WT constructs did not differ significantly following transfection in MCF-7 cells (P = 0.132) (Fig. 3B). However, a significant increase in reporter gene activity was observed following transfection of these constructs in HeLa cells (P = 0.013). These results indicate that, in HeLa cells, a cis-acting element(s) contained within the –2262 to –2151 conserved site is able to induce reporter gene transcription, approximately 60% above that of the full-length PTEN promoter alone. Interestingly, it has previously been reported that MCF-7 cells, along with other breast cancer cell lines, express USF but that, in these particular cells, this protein lacks transcriptional activity (29). Our observation that this conserved region failed to induce transcription in MCF-7 cells supports this report and also further indicates that USF proteins are involved in the transactivation of PTEN.


Figure 3
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  		Figure 3. The –2262 to –2151 conserved region is involved in transcriptional activation. (A) Schematic diagram of constructs used to assess the transcriptional activity of the –2262 to –2151 region: pGL3-B (empty vector), pGL3-B-FL (full-length PTEN promoter upstream of pGL3-B), pGL3-B-FL-2262-WT (–2262 to –2151 region upstream of the full-length PTEN promoter and pGL3-B) and pGL3-B-FL-2262-MT1 through pGL3-B-FL-2262-MT6 (constructs containing mutant –2181 to –2176 E-box/flanking sequences within pGL3-B-FL-2262-WT). (B) All three constructs were transiently cotransfected in either MCF-7 or HeLa cells along with the pRL-TK internal control plasmid and assayed for luciferase activity. Firefly luciferase measurements from three independent experiments were corrected for transfection efficiency using the Renilla luciferase internal control, averaged and then normalized to the full-length PTEN promoter construct (pGL3-B-FL). Error bars represent the standard deviation of three independent experiments. *P-value for pGL3-B-FL versus pGL3-B-FL-2262-WT in MCF-7 cells = 0.132. **P-value for pGL3-B-FL versus pGL3-B-FL-2262-WT in HeLa cells = 0.013. (C) Mutant reporter constructs (pGL3-B-FL-2262-MT1 through pGL3-B-FL-2262-MT6) were transfected into HeLa cells along with pRL-TK and assayed for firefly and Renilla luciferase activity. pGL3-B-FL-2262-MT1, pGL3-B-FL-2262-MT2 and pGL3-B-FL-2262-MT3 displayed a significant reduction in luciferase activity when compared with the wild-type construct (P ≤ 0.02). Similarly, pGL3-B-FL-2262-MT4 also resulted in a marked decrease. pGL3-B-FL-2262-MT5 and pGL3-B-FL-2262-MT6, did not differ significantly from the wild-type construct (P > 0.05).

 
We also generated six mutant reporter constructs, each containing mutations to either the –2181 to –2167 E-box consensus sequence or to its adjacent flanking sequence (Table 1 and Fig. 3A). All six constructs, along with the wild-type construct, were transfected into HeLa cells. pGL3-B-FL-2262-MT1, pGL3-B-FL-2262-MT2 and pGL3-B-FL-2262-MT3, which contain mutated half-sites or a completely mutated core, displayed a significant reduction in luciferase activity when compared with the wild-type construct (P ≤ 0.02) (Fig. 3C). Similarly, pGL3-B-FL-2262-MT4 also resulted in a marked decrease. pGL3-B-FL-2262-MT5 and pGL3-B-FL-2262-MT6, both of which contain alterations flanking the E-box motif, did not differ significantly from the wild-type construct (P > 0.05).

Together, these data further suggest that the –2181 to –2167 E-box is involved in the transactivation of PTEN and that this activity is mediated primarily by USF proteins.

USF1 and USF2, and not Myc/Max, bind the –2181 to –2176 PTEN E-box element
The absence of multiple shifted bands in Figures 1 and 2 suggests that a single DNA–protein complex is formed at the –2262 to –2151 site. Taken together with data demonstrating that the sequence flanking the –2181 to –2176 E-box element favors USF binding, this suggests that this DNA–protein complex is formed by USF protein binding. To examine this further, we performed mobility shift assays where antibodies raised against members of the bHLH-LZ family of transcription factors, specifically anti-USF1, anti-USF2, anti-Myc and anti-Max, were pre-incubated with either HeLa (Fig. 4A) or MCF-7 (Fig. 4B) nuclear extract. The USF protein complex can function either as a homodimer, consisting of either USF1 or USF2 dimers, or as a USF1–USF2 heterodimer, with the heterodimeric complex being predominant (30). Myc, on the other hand, exerts its transcriptional activity as a heterodimer in complex with Max (31). Because of this, each antibody was assayed singly as well as in their respective heterodimeric combination (i.e. anti-USF1/anti-USF2 and anti-Myc/anti-Max). The addition of anti-USF1, anti-USF2 and anti-USF1/anti-USF2 each resulted in a shift of the specific band formed by this DNA–protein complex in the presence of HeLa nuclear extract when compared with the complex formed in the absence of antibody (Fig. 4A, lanes 3, 4 and 5 versus lane 2). No shift was observed with the addition of anti-Myc, anti-Max, anti-Myc/anti-Max (Fig. 4A, lanes 6 through 8). As expected, a shift in the DNA–protein complex was not observed when anti-IgG was added to the reaction (Fig. 4A, lane 9). Similar results were observed in the presence of MCF-7 nuclear extract (Fig. 4B). These results demonstrate that in vitro, the native PTEN E-box allows for binding of the USF1 and USF2 proteins in both HeLa and MCF-7 nuclear extract.


Figure 4
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  		Figure 4. USF proteins, and not Myc/Max, bind to the –2262 to –2151 conserved region. SS-EMSAs were performed using the –2262 to –2151 region probe and antibodies against USF1, USF2, Myc, Max and IgG. (A) Lane 1: radiolabeled probe only (–), lane 2: radiolabeled probe plus HeLa nuclear extract (+). In lanes 3–9, radiolabeled probe, HeLa nuclear extract plus {alpha}-USF1, {alpha}-USF2, {alpha}-USF1/{alpha}-USF2, {alpha}-Myc, {alpha}-Max, {alpha}-Myc/{alpha}-Max and {alpha}-IgG antibodies were added, respectively. (B) Lane 1: radiolabeled probe only (–), lane 2: radiolabeled probe plus MCF-7 nuclear extract (+). In lanes 3–9, radiolabeled probe, MCF-7 nuclear extract, plus {alpha}-USF1, {alpha}-USF2, {alpha}-USF1/{alpha}-USF2, {alpha}-Myc, {alpha}-Max, {alpha}-Myc/{alpha}-Max and {alpha}-IgG antibodies were added, respectively.

 
Our analysis suggests that the USF proteins, and not Myc/Max, bind upstream of the PTEN promoter and transactivates its gene expression. However, the Myc–Max heterodimer can be difficult to assay in nuclear extract isolated from mammalian cells (32,33). Furthermore, Myc is a low abundant protein in many mammalian cells and tissues (34). Because of this, and to better understand which bHLH-LZ protein(s) binds the –2181 to –2176 E-box, we chose to further assess this region using USF1, Myc and Max recombinant proteins. USF1 specifically binds to the –2181 to –2176 E-box (lane 2 of Figs 5 and 6). Formation of this DNA–protein complex is abolished with the addition of excess specific competitor (lane 3 of Figs 5 and 6). Contrary to this, mutated competitor probes MT1, MT2 and MT3 do not compete with this binding reaction (Fig. 5, lanes 4, 5 and 6). The MT4 oligonucleotide probe, in which the two central nucleotides of the consensus E-box are inverted (CG to GC) is able to partially compete this binding, however, because USF1 has a higher affinity for the consensus sequence, a retarded band is observed (Fig. 5, lane 7). MT5 and MT6, both of which retain the consensus E-box element but contain mutated nucleotides in the flanking sequence to favor USF binding, are able to efficiently compete with the formation of this complex (Fig. 5, lanes 8 and 9). Moreover, the addition of anti-USF1 antibody to this reaction resulted in a super-shifted DNA–protein complex (Fig. 5, lanes 10 and 12), while the addition of the control anti-USF2 antibody did not (Fig. 5, lane 11).


Figure 5
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  		Figure 5. USF1 specifically binds to the –2181 to –2176 PTEN E-box element. EMSA and SS-EMSA assays were carried out using recombinant USF1 protein. Lane 1: radiolabeled probe only (–), lane 2: radiolabeled probe plus recombinant USF1 protein (+). In lanes 3–9, radiolabeled probe, recombinant USF1 protein, plus 100-molar excess of unlabeled specific competitor (Sp) and mutated competitor probes MT1 through MT6 were added, respectively. In lanes 10–12, SS-EMSAs were carried out using {alpha}-USF1, {alpha}-USF2 and {alpha}-USF1/{alpha}-USF2 antibodies, respectively.

 


Figure 6
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  		Figure 6. Myc/Max does not bind to the –2181 to –2176 PTEN E-box element. EMSAs were carried out using either recombinant USF1, Myc, Max or Myc/Max protein. Lane 1: radiolabeled probe only (–2262 to –2151) (–). Lanes 2, 4, 6 and 8 radiolabled probe (–2262 to –2151) were incubated using USF1, Myc, Max or Myc/Max recombinant protein, respectively (+). In lanes 3, 5, 7 and 9, these same reactions were carried out in the presence of 100-molar excess of unlabeled specific competitor probe (Sp). Lanes 10 and 16: radiolabeled Myc/Max consensus and Myc/Max mutant probes, respectively (–). Lanes 11, 12, 14, 17, 18 and 19 each were incubated with radiolabled probe (Myc–Max consensus: lanes 11, 12 and 14; Myc–Max mutant: lanes 17, 18, 19) and Myc, Max or Myc/Max recombinant proteins, respectively. In lanes 13 and 15, reactions in the presence of Max and Myc/Max recombinant proteins, respectively, were carried out in the presence of 100-molar excess of unlabeled Myc–Max consensus probe (Sp).

 
Next, the formation of a DNA–protein complex was assayed using Myc and Max recombinant proteins, both singly and in combination (i.e. Myc/Max) to interrogate formation of the Myc–Max heterodimer (Fig. 6, lanes 4–19). In contrast to the DNA–protein complex observed with USF1 recombinant protein (lane 2 of Figs 5 and 6), no complex was observed in the presence of recombinant Myc (Fig. 6, lanes 4 and 5). Interestingly, a faint, apparently specific band was observed in the presence of recombinant Max and Myc–Max proteins (Fig. 6, lanes 6–9), suggesting that these proteins may bind to the –2181 to –2176 E-box and compete for binding at this site. However, this interaction appears to have significantly less affinity than that observed with USF1. In addition, Max and Myc/Max protein were each able to bind to a radiolabeled Myc–Max consensus oligonucleotide probe (Fig. 6, lanes 12 and 14). Formation of this complex failed to occur in the presence of the radiolabeled Myc–Max mutant oligonucleotide probe (Fig. 6, lanes 18 and 19).

Taken together, these data provide further evidence that the highly conserved PTEN E-box is specifically and primarily bound by USF.

Mutation analysis of E-box region in CS/CS-like patients
To begin to assess the pathogenic role of the PTEN E-box element in CS, we analyzed this site along with 1.6 kb of flanking sequence (position –2895 to –1295) for germline genetic alterations in 30 previously identified germline PTEN mutation negative patients but with decreased PTEN protein levels and with classic CS (N = 15) or CS-like (N = 15) diagnostic features. No nucleotide variants were identified within the –2180 to –2176 E-box motif or within the adjacent sequence in these samples. Based on these data, we suspected that germline point mutations in the E-box consensus binding motif or within its adjacent flanking sequences are likely to be rare events in the CS and CS-like patients.

Deletions of PTEN have previously been identified only in BRRS which encompass the whole gene or localize to its 5' end, typically including exon 1 and often extending through exon 5 (3,35). To date, all germline deletions include at least exon 1 of PTEN. In other words, no deletions exclusively involving the highly conserved region upstream of PTEN, which includes both the PTEN full-length promoter and a putative CpG island (spanning position –2453 to –99), have been described. To assess whether deletions localize to this region, we performed real-time quantitative PCR in this same 30 PTEN mutation negative CS/CS-like patients. Copy number determinations were estimated for all samples, including five controls and one positive control sample with a known deletion, for regions targeting position –2237 to –2058 and PTEN exon 1 (c.52 to c.79+57). 2–{Delta}{Delta}Ct values among the control samples were 1.01 ± 0.09 and 1.00 ± 0.05 at each of the two regions, respectively, compared with 0.57 ± 0.05 and 0.60 ± 0.03 for the known deletion positive sample. Among the 30 CS/CS-like patients, three were found to have 2–{Delta}{Delta}Ct values suggestive of a hemizygous deletion at both the upstream E-box region and exon 1 (data not shown). One (11099–01) among 15 CS patients (7%) was found to be hemizygous only at the region encompassing the PTEN E-box element (0.52 ± 0.06 for the region spanning –2237 to –2058, compared with 0.89 ± 0.06 for exon 1) (Fig. 7A). Subsequent copy number determinations targeting PTEN exons 2 (c.80–51 to c.141, 2–{Delta}{Delta}Ct = 1.02 ± 0.08) and 5 (c.388 to c.492 + 40, 2–{Delta}{Delta}Ct = 1.06 ± 0.03) in this patient indicate that the entire PTEN gene proper is biallelic. Furthermore, we demonstrated that sample 11099–01 exhibited an increase in P-Akt, the downstream target of PTEN's lipid phosphatase activity, and a slight increase in P-p42/p44-MAPK, the target of PTEN's protein phosphatase activity, compared with controls (Fig. 7B). These data further suggest that aberrant regulation of PTEN is a manifestation of this deletion of the E-box region, affirming the integral role of this highly conserved upstream region in PTEN regulation, and points to a novel mechanism of disease etiology in CS patients.


Figure 7
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  		Figure 7. Identification of a functional hemizygous germline deletion upstream of the PTEN coding sequence. (A) Real-time quantitative PCR was used to investigate potential micro-deletions across the PTEN locus. Copy number determinations at position –2237 to –2058 and PTEN exons 1, 2 and 5 among five control samples were indicative of two alleles at each respective location (2–{Delta}{Delta}Ct = 1.00 ± 0.03 to 1.05 ± 0.12). One known deletion-positive sample displayed 2–{Delta}{Delta}Ct values consistent with a hemizygous deletion across all four regions (0.52 ± 0.04 to 0.60 ± 0.03). One PTEN mutation-negative sample exhibited 2–{Delta}{Delta}Ct values similar to those observed in the control samples for exons 1, 2 and 5 (0.89 ± 0.06 to 1.05 ± 0.03). In this same sample, copy number determinations targeting position –2237 to –2058 were indicative of a hemizygous deletion exclusively at this upstream region (2–{Delta}{Delta}Ct = 0.52 ± 0.06). (B) Western analysis reveals that the upstream deletion results in an increase in P-AKT levels, the downstream target of PTEN's lipid phosphatase activity, compared with normal control samples. In addition, P-p42/44-MAPK, the downstream target of PTEN's protein phosphatase activity, is also slightly increased in the sample harboring this deletion. Samples from normal control subjects (Controls 1 and 2), a known PTEN deletion positive sample (Del. positive), and the mutation negative sample with a novel deletion upstream of PTEN (11099–01) were assayed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Germline mutations in PTEN, the second most commonly mutated gene in all human cancers, are primarily associated with a number of clinically distinct heritable cancer syndromes, collectively referred to as PTEN Hamartoma Tumor Syndrome (PHTS) (2). PHTS is characterized by multiple hamartomatous lesions affecting derivatives of all three germ cell layers and includes both CS and BRRS (36). Germline PTEN mutations have been identified in 85% of patients diagnosed with CS and in 65% of patients diagnosed with BRRS (2,3). In a subset of classic CS patients lacking mutations in the PTEN coding sequence, approximately 10% were found to harbor mutations in the gene's core promoter (3). Furthermore, we also identified large germline hemizygous PTEN deletions, spanning the 5' region of PTEN and extending to its upstream region, in 11% of mutation-negative BRRS patients. All previously identified deletions have included at least one exon of PTEN. For the remaining 15 and 35% of CS and BRRS patients, respectively, the etiology of their disease remains unknown.

In order to better understand the mechanism(s) underlying the deregulation of PTEN in patients lacking mutations, in those with 5' deletions and mutations in the promoter, as well as those contributing to its phenotypic complexity, we sought to identify functional cis-regulatory elements involved in its regulation. We have previously shown that CS and BRRS patients lacking mutations in the PTEN gene and its core promoter are strongly associated with a haplotype block encompassing the region upstream of this gene and including a portion of its first intron (35). Because mutation scanning has failed to identify pathogenic mutations in these individuals, we hypothesized that other mechanisms potentially exist which can disrupt normal PTEN function and result in pathogenesis. Genetic alterations at cis-regulatory sites, either structural or functional in nature, could potentially alter the normal regulation of PTEN and, thus, represent one such mechanism.

Comparative genomic analysis has been shown to be a powerful method for identifying potential novel regulatory elements in genomic sequence. Using such an approach combined with cellular biology techniques, we demonstrate here that both USF1 and USF2 can bind to a highly conserved, novel PTEN E-box element. This element is located at position –2181 to –2176 relative to the PTEN translation initiation codon and within a conserved 102-bp fragment upstream of the PTEN core promoter. USF binding at the PTEN E-box element is both specific and functional, as we have demonstrated that, in cooperation with the full-length PTEN promoter, this region is able to mediate a 60% increase in reporter gene transactivation, compared with the full-length PTEN promoter alone. Interestingly, this response was only observed in HeLa cells and not in MCF-7 cells. This observation supports the findings of Ismail et al., who described the loss of USF transcriptional activity in several breast cancer cell lines, including MCF-7 cells, and provides additional evidence supporting the notion that the transactivation we observed was indeed mediated by USF proteins (29). Furthermore, mutation of the putative E-box site abolished its ability to bind USF. In addition, mutation of the –2181 to –2176 E-box core sequence in the pGL3-B-FL-2262 reporter construct results in a significant decrease in reporter gene activity compared to the wild-type construct.

USF1 and USF2 are ubiquitously expressed transcription factors comprised of highly conserved basic regions and both helix-loop-helix (HLH) and leucine zipper (LZ) domains (24,37,38). Residues forming the basic region enable USF proteins to bind DNA at consensus E-box motifs (CANNTG), while the HLH and LZ domains are primarily involved in its dimerization. Although several members of the bHLH-LZ family can recognize this motif, previous studies have shown that specific binding is dependent upon the flanking sequence surrounding the E-box site (25,27). Based on these studies, the sequence flanking the –2181 to –2176 E-box element appears to favor USF-specific binding. Through a series of EMSA experiments using mutant competitor probes designed in consideration of this evidence, we show that the –2181 to –2176 E-box is preferentially bound by USF. Moreover, using antibodies to specific bHLH-LZ proteins, SS-EMSAs further support these findings. Both USF1 and Myc/Max recombinant protein experiments provide clear evidence that, despite the redundancy with which proteins are able to bind to this motif, the DNA–protein complex formed at the –2181 to –2176 E-box is USF-specific.

USF proteins are involved in the transactivation of many human genes involved in a variety of cellular processes, including regulation of the cell cycle. In addition to regulating the expression of genes involved in the transition from G1/S and G2/M, including both cyclin B1 and Cdk1, USFs have also been shown to regulate known tumor suppressor genes, including p53, BRCA2 and APC (39–43). In the present study, we show that PTEN, a tumor suppressor phosphatase involved in regulating the cell cycle, is also transcriptionally regulated by USF.

p53 has been shown to inhibit USF-dependent BRCA2 promoter activation by preventing USF from binding to BRCA2's minimal promoter region, resulting in a decrease in both BRCA2 mRNA and protein levels (44). Although the precise mechanism by which p53 inhibits USF binding to the BRCA2 promoter has yet to be identified, p53's ability to regulate BRCA2's transcriptional activity suggests a regulatory loop among these important tumor suppressor genes. We and others have previously shown that similar co-regulation exists among p53 and PTEN (11,45–47). In addition to regulating PTEN gene expression, through the p53-binding site located in its promoter, PTEN and p53 also physically interact, forming a complex which serves to autoregulate PTEN's own expression. p53 and BRCA2's USF-mediated interaction suggests an additional mechanism by which p53 may regulate PTEN mRNA and protein expression.

A recent study by Nowak et al. described the involvement of PI3K in regulating USF-mediated transactivation of APOA5 (48). Together with our evidence suggesting that USF transactivates PTEN expression, this presents a potential self-regulatory mechanism involving the PI3K/Akt pathway. PTEN, the major 3-phosphatase in the PI3K/Akt pathway, antagonizes PI3K's activation of Akt by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate (PIP3) to phosphatidylinositol 4,5-bisphosphate (PIP2) (49). Phosphorylation of USF1 by PI3K prevents it from binding to the APOA5 E-box motif, thereby modulating its ability to transactivate APOA5 expression. Similarly, PI3K-dependent phosphorylation of USF1 could modulate binding at the PTEN E-box, resulting in PI3K-mediated transrepression of PTEN, however, this remains to be examined and is beyond the scope of the present study.

In the present study, we provide evidence suggesting that USF proteins regulate the PTEN tumor suppressor gene. The precise mechanism by which USF1 and USF2 regulate PTEN expression through the –2181 to –2176 E-box is unknown. USF1 has been shown to interact with other proteins involved in the assembly of the transcription preinitiation complex. Specifically, it has been described that USF1 can interact with TFII-I, an initiator-binding protein, and also bind the TFIID complex (50–52). Importantly, Roy et al. (53) also demonstrated that TFII-I can recruit the TFIID complex onto a TATA-less promoter. As has been postulated with the TATA-less promoter of APC, USF1 and USF2 may also interact through the TFII-I/TFIID complex at PTEN's TATA-less promoter and thereby facilitate its transactivation (42).

Based on previous studies describing USFs role in regulating other tumor suppressor genes and its anti-proliferative properties, deregulation of members of the USF protein family potentially has a role in tumorigenesis. USF genetic or epigenetic alterations may not only be important in carcinogenesis, but may also contribute to the phenotypic diversity observed in complex cancer syndromes. To date, however, no mutations have been described linking USF to cancer susceptibility. The only identified association has been with a familial combined hyperlipidemia (FCHL) risk haplotype involving two single nucleotide polymorphisms (SNPs) located on the USF1 locus (54). However, given their involvement in regulating cellular growth, genetic alterations in the USF genes may contribute to cancer susceptibility, either directly or perhaps through a modifying effect, by altering tumor suppressor gene regulation. The observation that cancer cell lines frequently lose USF transcriptional activity supports the potential role of their deregulation in carcinogenesis (29).

Given these data, we had hypothesized that alterations, whether structural or functional, at the PTEN E-box could contribute to altered PTEN expression and underlie disease in CS/CS-like patients with previously unidentified mutations and contribute to its phenotypic complexity. We have shown here that at least a subset of CS/CS-like patients without previously identified intragenic PTEN and promoter mutations harbor germline mutations of the E-box located more than 2 kb upstream of the gene's translation start site, but does not include its coding sequence. The pathogenicity is confirmed by an associated concomitant decrease in PTEN and upregulation of both downstream targets of PTEN's lipid and protein phosphatase activities. The CS patient with a germline hemizygous deletion of the E-box region, without involvement of PTEN or its promoter, has clinical features that include early onset breast cancer and macrocephaly. Interestingly, so far, germline promoter mutations in CS patients are strongly associated with breast pathology. Here, this patient with the E-box region deletion shows this similar phenotype.

Given PTEN's paramount role in both the cell cycle's pro-apoptotic pathway and in carcinogenesis, the intricacies of its transcriptional regulation necessitate further elucidation. We have identified a novel hexanucleotide E-box motif (CACGTG) located upstream of PTEN, which is specifically bound by the USF transcription factors USF1 and USF2 and is involved in the transactivation of PTEN. Using a multi-species comparative genomic approach, this study offers the first detailed look at evolutionarily conserved sequence elements along and flanking the PTEN locus and suggests that a comprehensive investigation of other conserved regions is warranted to better understand PTEN's complex regulatory pathways. Because there appears to be a range of transcription factor binding sites within and even upstream of PTEN's promoter region, as evidenced by this and previous reports, we can speculate that differential relative utilization of these different transcription factor binding sites, either because of structural variation or other unknown non-genetic mechanisms, can contribute to the diverse phenotypes of PHTS (3,35,46).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Comparative genomic analysis
Sequence data spanning 163 kb of the PTEN locus, to include the entire PTEN gene (103 kb) and 30 kb of flanking sequence, for Homo sapiens [chromosome 10, position 89 583 175–89 746 111, March 2006 Human Genome Assembly, NCBI Build 36.1 (hg18)] was obtained from the UCSC Genome Browser (http://genome.ucsc.edu/). Pair-wise sequence comparisons were carried out using the mVISTA software program (http://genome.lbl.gov/vista/index.shtml) for syntenic regions from Mus musculus [chromosome 19, position 32 793 494–32 916 025, February 2006 Mouse Genome Assembly (mm8)], and Rattus norvegicus [chromosome 1, position 236 741 027–236 867 261, November 2004 Rat Genome Assembly (rn4)] (55). For these comparisons, Homo sapiens was considered the base sequence and, prior to alignment, this sequence was masked for interspersed and simple repeat elements using the RepeatMasker software program (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker). A sliding window 100 bp in length was utilized to identify all contiguous subsegments that had a minimum sequence identity ≥70%.

Cell lines and culture
MCF-7 breast cancer cell lines and HeLa cervical cancer cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml each of penicillin and streptomycin and maintained at 37°C with 5% CO2. Nuclear protein extracts were isolated from MCF-7 cells with a high-salt method using NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer's protocol (Pierce, Rockford, IL, USA). Proteasome and phosphatase inhibitors were added to the extraction buffers in the following concentrations: 5 mg/ml aprotinin, 5 mg/ml leupeptin, 1 M PMSF, 1 M sodium orthovanadate, 1 M sodium fluoride, 1 M ß-glycerophosphate and 2.5 mg/ml pepstatin A.

Lymphoblastoid cell lines from two control samples and two CS patients with previously measured decreased PTEN protein (one with a known PTEN deletion and one with a newly identified deletion upstream of PTEN) were cultured in RPMI-1640 media supplemented with 20% FBS and 100 U/ml each of penicillin and streptomycin and maintained at 37°C with 5% CO2. Total protein extracts were then isolated using Mammalian Protein Extraction Reagent (M-PER; Pierce) supplemented with protease and phosphatase inhibitors.

Electrophoretic mobility shift assay (EMSA)
The 112 bp region spanning position –2262 to –2151 was subjected to PCR amplification using the following oligonucleotide primers containing EcoRI restriction sites (indicated in lowercase); forward 5'-GTCAgaattcCCCGAGCAAAGGAAGAAGAC-3' and reverse 5'-GTCAgaattcGTCGGAACTACTTTCCGAAG-3'. The resultant amplicon was digested with the EcoRI enzyme, purified and radiolabeled by incubation with Klenow fragment and alpha-32P dATP (3000 Ci/mmol, 10 mCi/ml). Two micrograms of either HeLaScribe (Promega, Madison, WI, USA) or isolated MCF-7 cell nuclear extract was then incubated with 1 ng of end-labeled DNA in binding buffer [10 mM HEPES (pH 7.9), 4% glycerol, 50 mM NaCl, 2.5 mM MgCl2, 0.5 mM DTT, 1µg/µl BSA, and 1µg poly dI-dC] for 20 min at room temperature. The resulting DNA–protein complexes were resolved on a 4% non-denaturing polyacrylamide gel for 3.5 h at 150 V at 4°C, dried and exposed to autoradiographic film with intensifying screens at –80°C. Specific and non-specific cold competitors, comprised of a 100-molar excess of unlabeled probe and an amplicon spanning exon 14 of PHLPP (an unrelated DNA fragment located on chromosome 18; forward 5'-TTGCATGCAAAGAGTAGGAG-3' and reverse 5'-TATGAATCCCATTGCCAGTG-3'), respectively, were added to subsequent reactions, as indicated, in order to determine binding specificity. Additional EMSAs were carried out using radiolabeled 68 bp amplicons generated using the following sets of primer pairs: (A) forward 5'-GTCAgaattcCCCGAGCAAAGGAAGAAGAC-3' and reverse 5'-GTCAgaattcGGAACTTTCCAAATTCCCAC-3' and (B) forward 5'-GTCAgaattcGGGAGTGGGAATTTGGAAAG-3' and reverse 5'- GTCAgaattcGTCGGAACTACTTTCCGAAG-3'. Nucleotides represented in lowercase indicate the EcoRI sites. A series of competition experiments were performed using a 100-molar excess of double-stranded cold oligonucleotide probes: specific oligonucleotide probe (–2200 to –2167, 34 bp) 5'-AAGTTCCCCAACTAGGGACACACGTGACCTCCTT-3', non-specific oligonucleotide probe (PTEN exon 5, 25 bp) 5'-GTAATGATATGTGCATATTTATTAC-3', Myc–Max consensus oligonucleotide probe 5'-GGAAGCAGACCACGTGGTCTGCTTCC-3' (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and Myc–Max mutant oligonucleotide probe 5'-GGAAGCAGACCACGGAGTCTGCTTCC-3' (Santa Cruz Biotechnology Inc.). As well, we constructed various mutant forms of the specific oligonucleotide probe (Table 1) and performed additional competition experiments. Subsequent EMSAs were also performed using recombinant USF1, Myc and Max protein (Protein One, Bethesda, MD, USA) as indicated in the corresponding figure legends.

For supershift EMSAs (SS-EMSAs), antibodies against USF1 (Santa Cruz Biotechnology Inc., sc-229), USF2 (Santa Cruz Biotechnology Inc., sc-862), Myc (Santa Cruz Biotechnology Inc., sc-764), Max (Santa Cruz Biotechnology Inc., sc-765) and IgG (Santa Cruz Biotechnology Inc., sc-2027) were obtained. SS-EMSA binding reactions were performed as described above with the following modification: for each reaction 2µg of antibody was pre-incubated with 4µg of nuclear extract for 20 min at 4°C prior to the addition of the appropriate radiolabeled probe and binding buffer. Following addition of the radiolabeled probe and binding buffer, the resulting reaction was continued for an additional 20 min at 4°C.

Luciferase gene reporter constructs and assays
The full-length PTEN promoter region located at position –1344 to –1 was PCR-amplified and subcloned upstream of the firefly luciferase gene and into the NheI/XhoI sites of the pGL3.1-Basic vector (Promega) (pGL3-B-FL) as previously described (46). In order to interrogate the conserved region located at position –2262 to –2151, an additional construct was made by inserting this region upstream of the full-length PTEN promoter in the pGL3-B-FL construct using the KpnI/NheI sites (pGL3-B-FL-2262-WT). The –2262 to –2151 insert was amplified from genomic DNA using the following primers: 5'-GTCAggtaccCCCGAGCAAAGGAAGAAGAC-3' and 5'-GTCAgctagcGTCGGAACTACTTTCCGAAG-3'. KpnI and NheI restriction sites, respectively, are indicated in lowercase for each primer. In addition, the GeneTailor Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA) was used to generate mutant reporter constructs containing mutated half-sites, core sequences or alterations in the flanking E-box motif (pGL3-B-FL-2262-MT1 through pGL3-B-FL-2262-MT6) (Table 1). All constructs were resequenced to confirm orientation and sequence integrity.

MCF-7 and HeLa cells were seeded using 1 ml DMEM in 12-well culture plates 24 h prior to transient transfection such that they were 50–60% confluent at the time of transfection. Cells were cotransfected with 0.5µg reporter construct and 10 ng pRL-TK Renilla luciferase vector (Promega) using 1.5 µl of FuGENE 6 (Roche Diagnostics, Indianapolis, IN, USA) for each transfection. pRL-TK Renilla luciferase activity was used to control for transfection efficiency. After 24–48 h post-transfection, cells were washed twice with PBS and harvested using passive lysis buffer as described by the manufacturer (Promega). Samples were analyzed for both firefly and Renilla luciferase activity by luminometry (Molecular Devices, Sunnyvale, CA, USA) using Dual-Luciferase Reporter Assay reagents according to the manufacturer's protocol (Promega) and normalized to Renilla luciferase expression. For each construct, three independent transfection experiments were performed.

Mutation and deletion analysis
Thirty patients (15 with a diagnosis of CS and 15 classified as ‘CS-like, i.e. had some features of CS but did not meet operational diagnostic criteria) without detectable germline PTEN mutations and with previously measured decreased PTEN protein expression were screened for genetic alterations at the PTEN E-box site. Briefly, PCRs were carried out using 12.5µl HotStarTaq Master Mix (Qiagen, Valencia, CA, USA), 10 mM forward primer (5'-TCTCAGCATTTCCGAATCAG-3'), 10 mM reverse primer (5'-CTGATGATGAAAGCTGAGATGG-3') and 20 ng of template DNA and used the following thermal cycling conditions: 95°C for 15 min, 34 cycles of 95°C for 30 s, 55°C for 45 s and 72°C for 2 min, followed by a 72°C final extension for 10 min. Purified PCR product was then subject to direct DNA resquencing (Lerner Research Institute, Genomics Core) and analyzed using Lasergene 7.0 software (DNASTAR Inc., Madison, WI, USA).

Real-time quantitative PCR was used to investigate potential micro-deletions in 30 PTEN mutation negative patient samples. Five normal control samples and one previously determined deletion positive sample (spanning both the PTEN and BMPR1A genes) were assayed as negative and positive controls, respectively. Copy number determinations were made for the region flanking the PTEN E-box (position –2237 to –2058, F: 5'-TGCCTCCGGAGCTATCACTG-3' and R: 5'-TACGGAACGGTAGGAAGCTG-3') and for exon 7 of a control reference gene, GAPDH (F: 5'-ATGCCTCCTGCACCACCAAC-3' and R: 5'-AGTCTTGGATGAGAAAGGTG-3'). Determination of gene copy number was assayed using 12.5µl SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 10 mM forward primer, 10 mM reverse primer and 20 ng of template DNA. Thermal cycling conditions comprised of 50°C for 2 min, 95°C for 10 min and 40 cycles at 95°C for 15 s followed by 58°C for 1 min using an ABI 7500 Sequence Detection System (Applied Biosystems). PCR efficiencies for each amplicon were determined by standard curve analysis using serial dilutions of genomic DNA from a control sample and ranged from 90–92% for these amplicons. Copy number determinations for PTEN exon 1 were performed as previously described (3). In addition, gene copy number was also assessed at PTEN exons 2 and 5 as previously described (35). Target and reference genes were assayed in duplicate for each sample and subject to meltcurve analysis and subsequent gel electrophoresis in order to determine amplicon specificity. Positive values were further assayed in at least two additional independent experiments. Gene copy number was determined using the comparative delta Ct method (2–{Delta}{Delta}Ct) as described by Livak et al. (56).

All subjects were enrolled by referral from centers throughout the United States, Canada and Europe following informed consent in accordance with the procedures approved by the human subjects protection committees of each respective institution. CS patients were classified in accordance with criteria established by the International Cowden Consortium and curated by the National Comprehensive Cancer Network (57).

Western analysis
Fifteen micrograms of protein from each sample was separated on a 10% SDS–PAGE gel, transferred to a nitrocellulose membrane and subsequently blocked for non-specific binding using 5% milk in 1% Tris-buffered saline containing 0.1% Triton X-100 (TBST). Membranes were incubated overnight with the following primary antibodies: phosphorylated p44/p42 MAPK (Cell Signaling, Danvers, MA, USA), phosphorylated Akt (Cell Signaling), both diluted 1:1000 in 3% BSA and ß-actin (diluted 1:5000 in 3% BSA; Sigma, St Louis, MO, USA). After incubation, membranes were washed with TBST, incubated with either anti-mouse IgG or anti-rabbit IgG secondary antibody (diluted 1:2500 in 5% milk; Promega), and the resulting protein bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Corp., Piscataway, NJ, USA).

Statistical analysis
All results are expressed as mean ± SD from three separate experiments. Statistical analysis was performed using Student's t-test and results are considered significant at the P < 0.05 level.


   ACKNOWLEDGEMENTS
 
This work was partially supported by The American Cancer Society (RSG-02–151-01-CCE to C.E.). C.E. is a recipient of the Doris Duke Distinguished Clinical Scientist Award. M.G.P. is a pre-doctoral fellow in the Cleveland Clinic Lerner Research Institute and also a graduate student of the Integrated Biomedical Sciences Graduate Program of The Ohio State University. C.E. is a Honorary Fellow of the Cancer Research UK Human Cancer Genetics Research Group, University of Cambridge, UK. M.G.P. would like to thank both Dr Yufang Tang and Ms Patricia Kessler for technical advice throughout the course of this study.

Conflict of Interest statement. The authors declare that they have no conflict of interest.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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