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[Cancer Research 66, 665-672, January 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

PEG10 Is a c-MYC Target Gene in Cancer Cells

Chi-Ming Li1, Adam A. Margolin1,2, Martha Salas1, Lorenzo Memeo3, Mahesh Mansukhani3, Hanina Hibshoosh3, Matthias Szabolcs3, Apostolos Klinakis1,4 and Benjamin Tycko1,3

1 Institute for Cancer Genetics, 2 Genome Center, and Departments of 3 Pathology and 4 Genetics and Development, Columbia University Medical Center, New York, New York

Requests for reprints: Benjamin Tycko, Institute for Cancer Genetics, Columbia University Medical Center, 1150 St. Nicholas Avenue, New York, NY 10032. Phone: 212-305-1149; Fax: 212-305-5489; E-mail: bt12{at}columbia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The product of the imprinted gene paternally expressed gene-10 (PEG10) has been reported to support proliferation in hepatocellular carcinomas, but how this gene is regulated has been an open question. We find that MYC knockdown by RNA interference suppresses PEG10 expression in Panc1 pancreatic carcinoma and HepG2 hepatocellular carcinoma cells and that knockdown of PEG10 inhibits the proliferation of Panc1, HepG2, and Hep3B cells. Conversely, PEG10 was up-regulated by inducing c-MYC expression in a B-lymphocyte cell line. Chromatin immunoprecipitation from Panc1 cells showed c-MYC bound to an E-box-containing region in the PEG10 first intron and site-directed mutagenesis showed that the most proximal E-box is essential for promoter activity. In a mouse mammary tumor virus (MMTV)-MYC transgenic mouse model of breast cancer, most but not all of the mammary carcinomas had strongly increased Peg10 mRNA compared with normal mammary gland. By immunohistochemistry, normal human breast and prostate epithelium was negative for the major isoform [reading frame-1 (RF1)] of PEG10 protein, but this cytoplasmic protein was strongly expressed in a subset of breast carcinomas in situ and invasive ductal carcinomas (~30%) and in a similar percentage of prostate cancers. As in the mouse model, we found positive, but not absolute, correlations between PEG10 and c-MYC in tissue arrays containing 161 human breast cancers (P < 0.002) and 30 prostate cancers (P = 0.014). Immunostaining of human placenta showed PEG10 and c-MYC proteins coexpressed in proliferating cytotrophoblast and coordinately lost in postmitotic syncytiotrophoblast. These findings link cancer genetics and epigenetics by showing that a classic proto-oncogene, MYC, acts directly upstream of a proliferation-positive imprinted gene, PEG10. (Cancer Res 2006; 66(2): 665-72)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cancer genetics and epigenetics have generally been studied as separate topics and unifying these research areas is an interesting challenge. It will be important to understand how genetic pathways intersect with epigenetic gene regulation in the multistage evolution of human cancers. The paternally expressed gene-10 (PEG10) gene was identified based on its location in an imprinted domain on human chromosome 7q21 and characterized as paternally expressed/maternally silenced (1). Peg10 is also imprinted in mice (2). The major open reading frame of PEG10 encodes a protein with distant homology to retroviral gag-pol proteins, suggesting that the gene arose from an ancient retroviral insertion event and then became fixed in mammalian evolution (1, 3, 4). Presumably related to its viral origin, the PEG10 mRNA encodes two protein isoforms (RF1 and RF2) via translational frameshifting (4). Recently, two groups have reported overexpression of PEG10 in hepatocellular carcinomas and in proliferating cells of regenerating normal liver, and using cell transfections, both concluded that this gene has growth-promoting activity (5, 6). PEG10 is also overexpressed in the embryonic form of biliary atresia, a disease associated with cell proliferation (7). Additional reports have suggested that PEG10 protein may act by blocking transforming growth factor ß (TGF-ß) signaling in epithelial cancers via binding to TGF-ß receptor II (8) or by blocking the apoptotic factor SIAH1 (5). It was recently found that specific chemokines induce PEG10 in normal B-lymphocytes and in B-cell leukemias, correlating with increased cellular resistance to apoptosis (9). However, the transcriptional pathways that activate this gene in proliferating cells have not been previously reported.

The c-MYC transcription factor heterodimerizes with MAX and activates a large number of downstream target genes, many of which are essential for cell growth and proliferation (10–12). In an expression profiling experiment in which we created a c-MYC knockdown in human carcinoma cells, we noticed that PEG10 was reproducibly down-modulated. Here we describe this experiment and additional analyses implicating c-MYC as an activator of PEG10 and showing recurrent acquisition of PEG10 protein expression in primary human breast and prostate cancers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Knockdown of c-MYC and PEG10 by RNA interference. Panc1 pancreatic carcinoma cells and HepG2 and Hep3B hepatocellular carcinoma cells were maintained in DMEM with 10% of fetal bovine serum plus 1.5 g/L sodium bicarbonate, 0.1 mmol/L nonessential amino acids, and 1 mmol/L sodium pyruvate. Two double-stranded short interfering RNAs (siRNA) directed to human c-MYC had the DNA target sequences 5'-AAGGACTATCCTGCTGCCAAG-3' (Qiagen-Xeragon, Germantown, MD) and 5'-AAGGTCAGAGTCTGGATCACC-3' (Qiagen-Xeragon); two siRNAs directed to human PEG10 had the DNA target sequences 5'-AAAGCTGGAGCGCTCCCACTA-3' (Qiagen-Xeragon) and 5'-AAGTCGCTGTCTGCTCTGATT-3' (Qiagen-Xeragon). Cells at 60% confluence on 60-mm tissue culture plates were transfected with 2 µg of the specific siRNAs or control siRNAs with scrambled DNA target sequence 5'-AATTCTCCGAACGTGTCACGT-3' using Transmessenger reagent (Qiagen, Valencia, CA). The transient transfection procedure was carried out twice on consecutive days. After another 24 hours of incubation, cellular RNA and protein were isolated and used for further analysis.

Gene expression profiling in Panc1 cells. Human genome 133A2.0 GeneChips (Affymetrix, Santa Clara, CA), which contain 22,000 oligonucleotide probe sets, querying 14,500 well-documented genes and 3,900 less well-characterized transcripts, were used for analyzing Panc1 RNAs from triplicate plates treated with MYC-directed or control siRNAs. The cRNA probes were synthesized as described (13). After scanning, mRNA expression values were determined using Affymetrix microarray suite v. 5.0 and significantly up-regulated and down-regulated genes were identified by ANOVA and displayed graphically using the GeneSpring software package (Agilent, Palo Alto, CA).

Induction of c-MYC and gene expression profiling in EREB.TMYC cells. EREB.TMYC B cells, which contain an EBV gene (EBNA-2) for conditional immortalization and a tetracycline-repressible c-MYC construct, were maintained in RPMI with 1 µmol/L ß-estradiol and 1 µg/mL tetracycline (14). Endogenous c-MYC expression was down-regulated due to growth arrest by removing ß-estradiol from the culture medium for 24 hours. After washing the cells with PBS, exogenous c-MYC expression was induced by incubating with medium lacking ß-estradiol and tetracycline. After 24 hours of induction, cellular RNA was harvested and used for expression profiling on U95A GeneChips (Affymetrix). Cells maintained in medium with tetracycline only for 48 hours were used as the c-MYC-negative control. The microarray data were analyzed as above.

Growth kinetics in RNA interference–treated cells. To measure the effect of PEG10 knockdown on cell growth, 2 x 105 of Panc1 cells, 1.5 x 105 of HepG2 cells, and 5 x 104 of Hep3B cells were plated in 60-mm dishes and, after 24 and 48 hours, were transfected with 1 µg of the specific siRNAs or control siRNAs. Cells from triplicate plates were counted in a hemocytometer at 72 hours and RNA harvested from parallel plates for Northern blot confirmation of the knockdowns.

Northern blot analysis of RNA and Southern blot analysis of DNA methylation. Total RNA was prepared after solubilizing cells or tissues in Trizol reagent (Invitrogen, Carlsbad, CA) according to the protocol of the manufacturer. The RNA was electrophoresed on 1.0% agarose gels containing formaldhehyde and then transferred to Nytran membranes (Schleicher & Schuell, Keene, NH). The blots were hybridized at 42°C in the Ultrahyb (Ambion, Austin, TX) solution, with cDNA probes specific for MYC or PEG10 (human and mouse probes for each gene were generated by reverse transcription-PCR; primers available on request), and washed at high stringency in 0.1% SDS and 0.1x SSC for 1 hour at 64°C. The blots were stripped and rehybridized with a probe for glyceraldhehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Genomic DNA was prepared by lysing the cells or tissue (pulverized under liquid nitrogen) in SDS/proteinase K, incubating for several hours at 50°C, followed by phenol/chloroform extraction and precipitation in ethanol. The DNA, 2.5 µg, was digested overnight with restriction enzyme RsaI, XbaI, CfoI, HpaII, or MspI (New England Biolabs, Beverly, CA), either singly or in the indicated combinations, and the digested DNA was resolved on 1% agarose gels, denatured, and neutralized under standard conditions and transferred to Nytran membranes. The Southern blots were hybridized with genomic probes specific to the human or mouse PEG10/Peg10 promoter regions. The primers for synthesizing the PEG10 promoter probe by PCR of human genomic DNA were PEG10 promoter US, 5'-CACGCAAAACTTGTCACGCC-3', and PEG10 promoter DS, 5'-GAGTGGGAGCCATTCCAAAAG-3'. The primers for generating the mouse Peg10 promoter probe were mPeg10 promoter US, 5'-CGAAGCACGCTGGGATTTGG-3', and mPeg10 promoter DS, 5'-GCTCTCGTCTCAGGATCTGG-3'.

Antibodies and Western blotting. After boiling at 100°C for 10 minutes in a denaturing solution containing 12 mmol/L Tris (pH 6.8), 5% glycerol, 0.4% SDS, 3 mmol/L 2-mercaptomethanol, and 0.02% bromophenol blue, 50 µg of total protein lysates from the experimental samples were electrophoresed on 4% to 20% polyacrylamide gradient/SDS gels (Invitrogen). After transferring to Immobilon membranes (Millipore, Bedford, MA) and blocking by 5% milk in 1x TBS, the membrane was hybridized with an affinity-purified rabbit polyclonal antibody against an 18-mer peptide at the COOH terminus of the major open reading frame (RF1; see ref. 4) of PEG10 (peptide sequence: N'-CPAKASKSSPAGNSPAPL-C'), or a mouse monoclonal antibody against {alpha}-actin (Sigma) as a loading control, in 1x TBS containing 5% dry milk and 0.1% Tween 20 overnight at 4°C. After washing, the signal was amplified and detected using a peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G (IgG; Amersham Pharmarcia Biotech, Piscataway, NJ) and enhanced chemiluminescence plus detection system (Amersham Pharmarcia Biotech).

Chromatin immunoprecipitation assays. Proliferating Panc1 cells, four 150-mm plates at 90% confluence, were analyzed using the ChIP-IT kit (Active Motif, Carlsbad, CA). Briefly, the cells were fixed in 1% formaldehyde at room temperature for 10 minutes and scraped in 1x PBS containing 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF) after treatment with glycine-containing stop-fix solution. After centrifugation, the cell pellet was resuspended and incubated for 30 minutes in ice-cold lysis buffer with PMSF and proteinase inhibitor cocktail. The nuclei were released by 10 strokes of an ice-cold Dounce homogenizer, centrifuged, and resuspended in shearing buffer. Chromatin was sheared to an average size of 1,000 bp (verified on an ethidium-stained agarose minigel) by sonication at 4°C. After preclearing with protein-G beads, in addition to saving an aliquot as a positive input control, the sheared chromatin was aliqouted into three tubes and incubated in ice-cold chromatin immunoprecipitation buffer for overnight at 4°C with 3 µg of polyclonal IgG antibody specific for c-MYC (N-262, Santa Cruz Biotechnology, Santa Cruz, CA), 3 µg of rabbit preimmune serum as a negative control (Santa Cruz Biotechnology), or 1.8 µg of transcription factor IIB antibody provided in the kit as a positive control, respectively. The immunoprecipitated chromatin was purified from the chromatin/antibody mixtures by incubating with protein G beads followed by several washing steps and the chromatin immunoprecipitation DNA was eluted in a solution containing 10 mmol/L NaHCO3 and 1% SDS. After removal of proteins by digestion with proteinase K, the DNA was purified and examined by PCR with primer pairs within and around the PEG10 promoter region. The primer sequences were A-forward: 5'-AAGCCTGAATGGAGTGTGCG-3', A-reverse: 5'-CAAAATACCCAGCCACCTTCTTC-3'; B-forward: 5'-CAACAGATTGTCAGTTTCCCAAGC-3', B-reverse: 5'-CGATTACGAGGTTTACACAGAGACC-3'; C-forward: 5'-CTCTGTTTTCTTGGAGCAGGACC-3', C-reverse: 5'-ACCACAACTACACCGCCACTTC-3'; D-forward: 5'-CTCCCTAAGCCTGCCTCTG-3', D-reverse: 5'-GGGCTCAGGCAAGGAAGGT-3'; and E-forward: 5'-TAACTTGTGCTGCCTCAGTCGC-3', E-reverse: 5'-CCTGCCACCAGACATTTCATTC-3'.

Promoter reporter assays and site-directed mutagenesis. The PEG10 promoter fragments, corresponding to nucleotides 80,003 to 81,049 of GenBank accession AC069292, were cloned upstream of the firefly luciferase gene in the pGL3-Basic vector (Promega, Madison WI) to yield the PEG10 promoter plasmid pGL3-PEG10. Three E-box elements in the first intron of PEG10, corresponding to nucleotides 81,698 to 82,061, 83,270, or 83,894 of GenBank accession AC069292, were sequentially cloned into the BamHI and SalI sites downstream of the luciferase cassette in the pGL3-PEG10 plasmid to yield the PEG10 promoter-reporter-enhancer plasmids pE1, pE12, and pE123, respectively. The mutant (cacatg -> cacaGT) construct at the first E-box of the PEG10 gene, pPEG10m, was generated by site-directed mutagenesis using the QuickChange kit (Stratagene, La Jolla, CA) with primers forward 5'-gcgctgcgaggcacaGTaactgcagaggtaca-3' and reverse 5'-tgtacctctgcagttACtgtgcctcgcagcgc-3'. The c-MYC expression construct, pMT2-MYC, and the E-box-containing construct, pTERT, and its E-box mutant construct, pTERTdm, used for the positive and negative controls in our experiments, have been previously described (15). To measure MYC responses, 7.5 x 104 Hep3B cells in 35-mm plates were transfected in triplicate with 0.2 µg of pTERT, pTERTdm, or each of the PEG10 promoter/E-box constructs with or without 0.2 µg of pMT2-MYC using Lipofectamine 2000 transfection reagent (Invitrogen). As controls for normalization, 50 ng of the Renilla luciferase plasmid, pTK-luc (Promega), and 50 ng of the green fluorescent protein plasmid, pEGFP-N1 (Clontech, Mountain View, CA), were cotransfected and dual luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) after 36 hours. All transfections were done in triplicate.

Immunohistochemistry for PEG10. Formalin-fixed, paraffin-embedded sections of human breast carcinomas, including adjacent nonneoplastic glandular structures, atypical ductal hyperplasia and carcinoma in situ lesions, and custom breast cancer and prostate cancer tissue arrays produced in the Columbia University Pathology Department, were heated in 10 mmol/L citrate buffer (pH 6.0) in a microwave oven at the maximum power for 10 minutes and then at a reduced power for 15 minutes for antigen recovery, and the sections were blocked in 5% or 10% goat serum for 20 minutes and then exposed to affinity-purified antiserum raised against a COOH-terminal peptide of PEG10 (see Western blotting methods above) at a 1:200 dilution in 1x PBS containing 0.01% Tween 20 for 1 hour at room temperature or to an anti-c-MYC antibody (N-262; Santa Cruz Biotechnology) at a dilution of 1: 200 for overnight at 4°C. The sections were then incubated with the secondary antibody, goat anti-rabbit IgG, at 1:200 dilution in 1x PBS containing 0.01% Tween 20 for 30 minutes and developed using the Elite ABC kit (Vector Laboratories, Burlingame, CA). The sections were counterstained with hematoxylin and microscopic images were captured digitally. The results for c-MYC and PEG10 immunostaining in breast cancers were scored independently and blindly by three surgical pathologists (H.H., L.M., and M.S.) who concurred in the scoring. The results for c-MYC and PEG10 in prostate cancers were scored independently and blindly by two surgical pathologists (M.M. and M.S.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RNA interference–mediated c-MYC knockdown suppresses PEG10 expression in carcinoma cells. To identify candidate c-MYC target genes in Panc1 pancreatic carcinoma cells, we transfected these cells with MYC-directed or control siRNAs and profiled gene expression using oligonucleotide microarrays. The RNA interference procedure effectively suppressed both MYC mRNA and c-MYC protein expressions (Figs. 1A and 2). This c-MYC knockdown was accompanied by cell growth arrest without loss of cell viability. Among the large number of genes scoring as significantly down-modulated in the microarray data, we noticed an imprinted gene, PEG10 (Fig. 1B). This gene was consistently suppressed by MYC siRNA with down-modulation seen not only in Panc1 cells but also in the hepatocellular carcinoma line HepG2 (Fig. 2A). The effect was validated using two double-stranded siRNAs, matching different sequences within the MYC transcript (see Materials and Methods), arguing against "off-target" artifacts. Interestingly, the SGCE gene, which is oriented head-to-head with PEG10, separated by an 800-bp CpG island, and is coordinately imprinted in the same parental "direction" with PEG10, was not affected by the c-MYC knockdown. Because of our interest in pathways involving imprinted genes, we selected the PEG10 gene for further validation as a c-MYC target.


Figure 1
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  		Figure 1. Knockdown of c-MYC protein by MYC-directed siRNA down-modulates PEG10 mRNA and activation of c-MYC induces PEG10 mRNA. A, MYC-directed and scrambled (S) control siRNAs were introduced into Panc1 cells by two successive transient transfections (see Materials and Methods). Cellular proteins were immunoblotted with antibody against human c-MYC. Immunoblotting for ß-actin is shown as a loading control. B, cRNA probes from four replica plates of Panc1 cells transfected with scrambled siRNA and four plates of Panc1 cells transfected in parallel with MYC-directed siRNA were applied to Affymetrix 133A GeneChips. Genes (probe sets) differentially expressed between these two conditions were identified by three successive filtering steps: first, ANOVA requiring P < 0.01; second, requiring Affymetrix presence calls in at least three of the eight samples; and third, requiring 1.3-fold variation from the experiment mean in at least three of the eight samples. This procedure produced 79 probe sets reporting increased mRNA expression (blue lines) and 248 probe sets reporting decreased mRNA expression (red lines) after MYC RNA interference. Solid, dashed, and stippled white lines, mRNA values for PEG10, MYC, and SGCE, respectively, normalized to the experiment mean. C, cRNA probes from six replica plates of EREB.TC-MYC cells in the presence of tetracycline (c-MYC negative) and six replica plates in the absence of tetracycline (MYC positive) were hybridized to Affymetrix U95A GeneChips. The data were filtered as in (B).

 

Figure 2
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  		Figure 2. Half-life of MYC and PEG10 mRNAs and effects of PEG10- and MYC-directed RNA interference on cell proliferation. A, MYC-directed RNA interference down-modulates PEG10 mRNA in both Panc1 and HepG2 carcinoma cells whereas PEG10-directed siRNA effectively reduces PEG10, but not MYC mRNA. The lanes correspond to duplicate transfections for each condition. B, half-life determination for MYC and PEG10 mRNAs. Total RNA was analyzed by Northern blotting at the indicated times after the addition of actinomycin D (5 µg/mL). An ethidium bromide–stained image of the 28S rRNA is shown as a loading control. PEG10 mRNA is seen as a major band at ~6 kb and a minor band at a larger size. C, PEG10-directed siRNA slows the growth of pancreatic and hepatocellular carcinoma cells. Cells (triplicate plates) were transfected at 24 and 48 hours with the indicated siRNAs and cell counts were determined at 72 hours. Columns, percent increase in cell number between 24 and 72 hours; bars, SD.

 
Activation of c-MYC induces PEG10 mRNA in conditionally immortalized B cells. To ask whether the converse situation, activation of PEG10 transcription by inducing c-MYC expression, could also be shown, we examined oligonucleotide microarray data from EREB.TC-MYC cells, a conditionally immortalized B-cell line in which cell proliferation and MYC expression could be controlled independently. In this cell line, immortalization is dependent on induction by estrogen because the EBV gene encoding EBNA-2 is expressed as a chimeric fusion with the hormone-binding domain of the estrogen receptor and c-MYC protein expression can be induced by removing tetracycline from the culture medium because this line carries a TET-repressible c-MYC expression construct. In these cells, we observed strong activation of PEG10 mRNA at 24 hours after induction of exogenous c-MYC by the removal of tetracycline (Fig. 1C). In contrast, SGCE mRNA was not induced in these cells and in fact was slightly repressed after c-MYC induction (Fig. 1C), a result paralleling the negative findings for this flanking gene in our c-MYC knockdown experiments.

Knockdown of PEG10 by RNA interference inhibits carcinoma cell proliferation. MYC mRNA is known to turn over rapidly in the cell, allowing the levels of c-MYC to be regulated during the cell cycle. When we inhibited new mRNA synthesis in Panc1 cells using actinomycin D and measured PEG10 and MYC mRNA over time, we found that PEG10 mRNA was stable for up to 8 hours whereas MYC mRNA declined by 1 hour and disappeared from the cells by 2 hours (Fig. 2B). Despite this stability of PEG10 mRNA under normal conditions, the levels of this transcript were strongly reduced by transfecting cells with PEG10-directed siRNA (Fig. 2A). Because prior data have suggested a proliferation-positive function for the PEG10 gene, we asked whether down-modulating PEG10 expression by RNA interference would inhibit the growth of a series of carcinoma cell lines [i.e. Panc1, HepG2, and Hep3B (hepatocellular carcinoma)]. We found that cell proliferation was significantly inhibited in each of these lines but that in each cell line the PEG10 knockdown had a weaker inhibitory effect than a MYC knockdown (Fig. 2C). By using two different PEG10-directed siRNAs, we confirmed that the inhibition of cell proliferation was a specific effect due to the PEG10 knockdown. These data are consistent with PEG10 being one of many effector genes influencing net cell proliferation downstream of MYC.

c-MYC protein binds to E-box-containing sequences in the PEG10 first intron. Analysis of the human PEG10 genomic sequence revealed three consensus E-boxes (CACATG) in the first intron (Fig. 3A). To determine whether the PEG10 promoter region is a direct target for the c-MYC transcription factor, which binds E-box sequences as MYC/MAX heterodimers, we carried out chromatin immunoprecipitation assays. Proliferating Panc1 cells were treated with formaldehyde to produce protein-DNA cross-links and chromatin immunoprecipitation was done using anti-c-MYC or control preimmune serum. We queried five regions of the DNA in and around the PEG10 gene by PCR of the immunoprecipitated DNA. As shown in Fig. 3A, this procedure revealed c-MYC protein bound specifically to DNA near the PEG10 E-box sequences but not to more distant upstream or downstream sequences.


Figure 3
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  		Figure 3. PEG10 is a direct transcriptional target of c-MYC. A, chromatin immunoprecipitation assays showing c-MYC binding to an E-box-containing region in PEG10 intron 1. The genomic DNA around PEG10 is approximately drawn to the scale; rectangles, first exons of SGCE and PEG10. These genes are divergently transcribed (arrows). Closed circles, E-boxes, CAC(G/A)TG. For the chromatin immunoprecipitation assays, genomic DNA from Panc1 cells was cross-linked and sonicated, then immunoprecipitated with a rabbit polyclonal antibody against human c-MYC (MYC) or with control preimmune serum (PI). After reversing the cross-links, PCR was done to map the region bound by c-MYC using the five indicated primer pairs. The resulting 200- to 300-bp PCR products are shown. Regions C and D bind c-MYC. Input DNA (0.5% of the total chromatin DNA; I) was the positive control for each PCR reaction. Similar results were obtained in three experiments. B, site-directed mutagenesis of the PEG10 promoter. The indicated promoter fragment containing the proximal intronic E-box has MYC-responsive activity in the luciferase reporter assay, which is abrogated by a point mutation in the E-box. The MYC construct, pMT2-MYC, used to ascertain E-box-dependent MYC/MAX activity, the E-box-containing construct, pTERT, and its E-box mutant construct, pTERTdm, used as positive and negative controls, have been previously described (15). See Materials and Methods for details of the plasmid constructs.

 
As noted above, the expression of the SGCE gene, which is the closest flanking gene to PEG10 and which is co-coordinately imprinted with PEG10, was not strongly affected by up- or down-modulation of c-MYC, and in fact was slightly repressed by activation of c-MYC in the B cell line. This gene specificity of the regulation of PEG10 by c-MYC may be facilitated by the fact that the binding sites for c-MYC are located in the PEG10 first intron and not in the 800-bp CpG island between SGCE and PEG10.

The proximal intronic E-box is essential for PEG10 promoter activity in reporter assays. To further test whether PEG10 is a direct c-MYC target gene, we cloned a nested series of PEG10 intron fragments downstream of the firefly luciferase (LUC) gene, placing the PEG10 5' sequences (the region between PEG10 and SGCE) upstream of LUC. This series of promoter-reporter-enhancer plasmids allowed us to test which of the three E-box elements in the first intron were crucial for PEG10 promoter activity in Hep3B cells. Luciferase assays showed that the smallest construct containing only the most proximal E-box (363 bp of first intron sequence) had full activity, with no increase in reporter expression seen with the longer inserts containing both the proximal E-box and the two downstream E-boxes (data not shown). We therefore carried out site-directed mutagenesis of this minimal promoter-reporter-enhancer construct, changing the E-box CACATG sequence to CACAGT (Fig. 3B). This mutation completely abolished basal promoter activity and abrogated the response to exogenous c-MYC (Fig. 3C). These findings suggest that the E-box in the PEG10 first intron is an essential promoter/enhancer element. In conjunction with the chromatin immunoprecipitation data above, these results implicate PEG10 as a direct transcriptional target of c-MYC.

The mouse Peg10 gene is frequently activated in c-MYC-driven mammary carcinomas. The sequence of PEG10 is conserved (66% amino acid identity) between humans and mice, as is the gene structure, suggesting that it should be possible to study the regulation of this locus in cancer using mouse model systems. Mice carrying an mouse mammary tumor virus (MMTV)-MYC transgene have a highly penetrant phenotype of mammary carcinoma, with most of the animals developing palpable tumors by around 8 months (16, 17). To ask whether the murine Peg10 gene is a target of c-MYC in tumorigenesis, we collected biopsies of 20 mammary carcinomas arising in these mice and measured Peg10 mRNA in these tumors and in two control biopsies of normal mammary glands by Northern blotting and Phosphorimaging. Peg10 mRNA was expressed at very low levels in the normal mammary gland, with Northern blot bands visualized only after prolonged exposures of the blots. In contrast, of the 20 mammary carcinomas, 11 expressed easily detectable Peg10 mRNA, substantially elevated compared with the normal mammary tissue (Fig. 4A and B), with 7 of these tumors showing a >10-fold increase (Fig. 4B).


Figure 4
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  		Figure 4. Peg10 expression is activated in breast carcinomas from MMTV-MYC transgenic mice. A, total RNA from primary breast tumors from MMTV-MYC transgenic mice was analyzed by Northern blotting with Peg10 and MYC probes. The tumors overexpress Peg10 mRNA relative to the normal mammary gland. GAPDH is a loading control. B, phophorimaging data from Northern blots analyzing 22 mammary tumors and two normal multiparous (nonlactating) mammary glands. The tumors are grouped based on their MYC mRNA expression, with low indicating the few tumors with transgenic MYC mRNA bands not detectable after 3-day exposure of Northern blots and high indicating the majority of the tumors with transgenic MYC mRNA clearly seen on Northern blots after overnight exposure. Filled diamonds, tumors showing Peg10 promoter hypermethylation by Southern blotting; open diamonds, tumors and normal controls with normal methylation. C, analysis of CpG methylation in the mouse Peg10 promoter by Southern blotting. Genomic DNA from MMTV-MYC tumors (T) and normal mammary gland (N) was digested with RsaI (R), either alone or with the methylation sensitive restriction enzymes CfoI (C) or HpaII (H). MspI (M) is a non-methylation-sensitive isoschizomer of HpaII. A restriction map of the Peg10 promoter region is shown below with the flanking RsaI sites scored by the bold lines and the internal CfoI and HpaII sites scored by thin lines. Filled circles, E-boxes. The Peg10 CpG island is biallelically methylated (protected from CfoI and HpaII digestion) in tumor T10 but shows a normal imprinted pattern in the other tumor (T2), with one allele methylated and therefore not digested (dash) and the other allele unmethylated and therefore digested (brackets).

 
Although all of the carcinomas in this mouse model are initiated via overexpression of the MYC transgene, rehybridization of the Northern blots with a MYC probe showed that as these tumors progress, some of them cease to express high levels of MYC mRNA. Consistent with Peg10 being a target of c-MYC in this system, Peg10 mRNA was expressed very poorly in both of the two carcinomas that no longer expressed MYC (Fig. 4B). However, we also observed a third class of tumor in which Peg10 mRNA was poorly expressed despite persistent high expression of MYC. Among these seven tumors, Southern blotting of genomic DNA digested with methylation-sensitive restriction enzymes showed that the Peg10 promoter was hypermethylated at CpG sites in one case whereas the remaining six Peg10-low cases did not show promoter hypermethylation (Fig. 4C and data not shown). An additional example of Peg10 promoter hypermethylation was found in one of the two tumors with low MYC mRNA. The methylation of the Peg10 promoter was not affected in the tumors with high expression of Peg10, which retained a biphasic pattern (fully methylated and unmethylated bands corresponding to the imprinted and nonimprinted alleles, respectively) identical to the normal mammary gland. In aggregate, these data indicate that Peg10 is frequently activated in this c-MYC-driven transgenic model of breast cancer without loss of methylation imprinting, and that in some of the mammary tumors this gene can be a target for epigenetic repression, either with or without a gain of promoter methylation.

To ask whether MYC expression is sufficient for inducing Peg10 in the mouse mammary gland, we examined nonneoplastic mammary glands from first pregnancy MMTV-MYC mice by Northern blotting. Peg10 mRNA was not highly expressed in these nonneoplastic glands although MYC mRNA was expressed at levels comparable to those seen in the breast tumors (data not shown). Overall, these results suggest that additional factors associated with the transition to neoplasia are necessary to activate Peg10 and that c-MYC is necessary, but not sufficient, for Peg10 induction in this model system.

PEG10 is activated in subsets of human breast and prostate carcinomas. A substantial subset of human breast carcinomas, including ductal carcinoma in situ, overexpress MYC either due to gene amplification or transcriptional deregulation (18–20). To ask whether the PEG10 gene is activated in primary human breast cancers, we carried out immunohistochemistry for PEG10 protein, using an affinity-purified polyclonal antiserum raised against a peptide epitope in the PEG10 major open reading frame (the RF1 protein isoform), in a series of 23 paraffin-embedded breast biopsies with standard histologic sections and in a second series of 161 breast cancer cases in a tissue array. Several of the cases examined in standard sections contained areas of ductal carcinoma in situ and invasive ductal carcinoma, as well as nonneoplastic glandular tissue, thus allowing an assessment of PEG10 protein expression during breast cancer progression. The results indicated that PEG10 is not detectable in normal quiescent breast epithelial cells but that this protein becomes strongly expressed in 55% of ductal carcinoma in situ lesions and 32% of invasive ductal carcinomas, where it is found predominantly in the cytoplasm (Table 1A; Fig. 5A-F). In the tissue array, 36% of the breast carcinomas, which were mostly invasive ductal cancers with a small number of ductal carcinoma in situ lesions, were PEG10 positive (Table 1B). Combining the standard sections and the tissue array, we found 11 PEG10-positive cases in which both a ductal carcinoma in situ and an invasive component could be evaluated, and in 8 of these cases, immunoreactive PEG10 was either restricted to the ductal carcinoma in situ or was more strongly expressed in the ductal carcinoma in situ compared with the invasive carcinoma (Fig. 5D-F). As shown in Table 1B, results from serial sections of the tissue array stained with anti-PEG10 and anti-c-MYC antibodies showed that PEG10 protein expression is positively (P < 0.002, Fisher's exact test), but not absolutely, correlated with c-MYC expression in human breast cancers.


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  		Table 1. Immunostaining for PEG10 and c-MYC in human breast and prostate cancers

 

Figure 5
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  		Figure 5. PEG10 protein (RF1) expression in human breast and prostate carcinomas and in placental cytotrophoblast. Histologic sections from one patient with breast cancer are shown in (A-C) and from another patient in (D-F), immunostained with affinity-purified antipeptide antiserum recognizing PEG10 RF1. The nonneoplastic breast epithelium (A and D) is negative for PEG10 in both cases whereas the carcinoma in situ lesions (B and E) are positive. The invasive carcinoma remains positive in the first patient (C) but is negative for PEG10 in the second patient (F). A section of a PEG10-positive prostate carcinoma is shown in (G); the nonneoplastic prostate gland (N) is only faintly positive for PEG10 (in basal epithelial cells); the benign lymphoid infiltrate (Ly) is negative; and the adenocarcinoma cells (CaP) are strongly positive for cytoplasmic PEG10 at the luminal borders of the malignant glands. The numbers of PEG10-positive and PEG10-negative cases and correlations with immunoreactive c-MYC protein are shown in Table 1A to C. H and I, immunohistochemistry for c-MYC (H) and PEG10 (I) in human placenta. Arrows, proliferating villous cytotrophoblast layer, which is PEG10 and c-MYC positive; asterisks, overlying layer of postmitotic syncytiotrophoblast, which is PEG10 and c-MYC negative.

 
Similar results were obtained with a tissue microarray containing 30 prostate carcinomas. The normal prostatic epithelium stained only very weakly for the RF1 isoform of PEG10, and only in the basal cell layer, whereas 37% of the prostate cancers in this tissue array were strongly PEG10 positive (Fig. 5G). As was true for the breast carcinomas, we observed a positive, but not absolute, correlation of PEG10 with c-MYC in these prostate cancers (Table 1C).

PEG10 and c-MYC are coexpressed in placental cytotrophoblast. Lastly, we wished to determine if there is a correlation between PEG10 and c-MYC protein expressions in a major normal site of PEG10 expression, the developing placenta. We carried out immunostaining of a normal mid-gestation (16 weeks) placenta, applying anti-PEG10 and anti-c-MYC to serial sections of the chorionic villi. These villi are organized as three major tissue layers from inside to outside: a mesenchymal core, a cytotrophoblast layer, and a syncytiotrophoblast layer. The cytotrophoblast is a proliferating tissue compartment, which is the direct precursor of the postmitotic syncytiotrophoblast. We found that nuclear c-MYC and cytoplasmic PEG10 were coexpressed in the cytotrophoblast layer whereas both gene products were absent from the overlying syncytiotrophoblast (Fig. 5H and I). Also evident in these tissue sections was the more uniform expression of PEG10 compared with c-MYC (Fig. 5H and I). This finding is consistent with the rapid regulation of the MYC gene in the cycling cells of this growing tissue and the greater stability of PEG10 mRNA than MYC mRNA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Certain imprinted genes play a role in the initiation and progression of human cancers and this in part reflects a subversion of the normal functions of these genes in regulating tissue growth. Epigenetic phenomena such as de novo DNA methylation and loss of imprinting have been well documented in preneoplastic and malignant tissues (21) but the problem of how classic genetic pathways may contribute to the dysregulated expression of imprinted genes has received less attention. In this report, we describe a link between the classic proto-oncogene MYC and the proliferation-positive imprinted gene PEG10. PEG10 is imprinted in humans and mice, with the paternal allele active and maternal allele silent. PEG10 encodes a protein with distant similarity to retroviral gag-pol proteins and is expressed most strongly in the developing placenta but also in certain normal adult organs including the heart and lung (2, 3). The PEG10 gene is activated in a variety of human cancers and data from manipulating the expression of this gene in cancer cell lines, both in prior work (5, 8) and in the current study, indicate that it has a positive role in cell proliferation. Given the parental "direction" of its imprinting (paternal allele active), this growth-positive role for PEG10 would be predicted by the genomic conflict or kinship theory of imprinting (22).

With this background, we were intrigued by finding PEG10 as a c-MYC-responsive gene in our MYC RNA interference experiments, and as shown here, we have validated this gene as a direct downstream target of c-MYC by additional functional, biochemical, and correlative criteria (i.e., induction after c-MYC activation in EREB.TC-MYC cells, chromatin immunoprecipitation with anti-c-MYC, E-box-dependent promoter activity in reporter assays, and positive correlations with c-MYC expression in a mouse model of breast cancer and in human primary breast and prostate carcinomas). The positive correlation of cytoplasmic PEG10 protein with nuclear c-MYC in a specific tissue compartment of the normal human placenta further supports PEG10 as a c-MYC target gene. How PEG10 protein acts in the cytoplasm to promote or support cell proliferation is not yet certain but data have been reported for two candidate pathways—inhibition of apoptosis and inhibition of TGF-ß signaling (5, 8, 9).

We have also shown evidence for several factors that could explain the minority of primary cancers with discordant expression of PEG10 and MYC. Epigenetic silencing of Peg10 by promoter methylation can explain some cases of MMTV-MYC-induced mammary carcinomas that fail to express Peg10 mRNA whereas other cases must reflect non-methylation-dependent silencing. In the future, it will be interesting to study whether the loss of Peg10 expression in this group of mouse carcinomas is analogous to the loss of expression of this gene that we have described in the invasive component of some human breast cancers in which the intraductal component is PEG10 positive. There are precedents for cancer-associated genes that are subject to different selective pressures in early (in situ) versus late (invasive) tumors, a notable example being genes in the TGF-ß pathway. In a situation opposite to PEG10, these TGF-ß pathway genes are selected for loss of function at early stages but gain of function at late stages in the evolution of human carcinomas. A factor that we can propose to explain some of the human cancers that are PEG10 positive/c-MYC negative by immunohistochemistry is the long half-life of PEG10 mRNA in the cell, contrasting with the short half-life of MYC mRNA. In addition, there are likely to be parallel pathways for activating PEG10, possibly via other E-box binding proteins. Lastly, the antiserum used in this study recognizes only PEG10 RF1, and in the future it will be interesting to characterize the expression in cancer cells of the alternative isoform of this protein (RF2), which can be produced from the PEG10 mRNA transcript by translational frameshifting (4).


   Acknowledgments
 
Grant support: Department of Defense grant BC030061 (B. Tycko).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Michelle Wei for affinity purification of anti-PEG10, Rongzhen Chen for PEG10 immunohistochemistry, and Katia Basso and Brendan Chen in the Dalla-Favera laboratory for helpful advice.

Received 5/ 5/05. Revised 10/13/05. Accepted 11/ 2/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ono R, Kobayashi S, Wagatsuma H, et al. A retrotransposon-derived gene, PEG10, is a novel imprinted gene located on human chromosome 7q21. Genomics 2001;73:232–7.[CrossRef][Medline]
  2. Ono R, Shiura H, Aburatani H, Kohda T, Kaneko-Ishino T, Ishino F. Identification of a large novel imprinted gene cluster on mouse proximal chromosome 6. Genome Res 2003;13:1696–705.[Abstract/Free Full Text]
  3. Smallwood A, Papageorghiou A, Nicolaides K, et al. Temporal regulation of the expression of syncytin (HERV-W), maternally imprinted PEG10, and SGCE in human placenta. Biol Reprod 2003;69:286–93.[Abstract/Free Full Text]
  4. Manktelow E, Shigemoto K, Brierley I. Characterization of the frameshift signal of Edr, a mammalian example of programmed –1 ribosomal frameshifting. Nucleic Acids Res 2005;33:1553–63.[Abstract/Free Full Text]
  5. Okabe H, Satoh S, Furukawa Y, et al. Involvement of PEG10 in human hepatocellular carcinogenesis through interaction with SIAH1. Cancer Res 2003;63:3043–8.[Abstract/Free Full Text]
  6. Tsou AP, Chuang YC, Su JY, et al. Overexpression of a novel imprinted gene, PEG10, in human hepatocellular carcinoma and in regenerating mouse livers. J Biomed Sci 2003;10:625–35.[Medline]
  7. Zhang DY, Sabla G, Shivakumar P, et al. Coordinate expression of regulatory genes differentiates embryonic and perinatal forms of biliary atresia. Hepatology 2004;39:954–62.[CrossRef][Medline]
  8. Lux A, Beil C, Majety M, et al. Human retroviral gag- and gag-pol-like proteins interact with the TGF-ß receptor ALK1. J Biol Chem 2004;280:8482–93.[Medline]
  9. Hu CS, Xiong J, Zhang LJ, et al. PEG10 activation by co-stimulation of CXCR5 and CCR7 essentially contributes to resistance to apoptosis in CD19+CD34+ B cells from patients with B cell lineage acute and chronic lymphocytic leukemia. Cell Mol Immunol 2004;1:280–94.[Medline]
  10. Grandori C, Cowley SM, James LP, Eisenman RN. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu Rev Cell Dev Biol 2000;16:653–99.[CrossRef][Medline]
  11. Fernandez PC, Frank SR, Wang L, et al. Genomic targets of the human c-Myc protein. Genes Dev 2003;17:1115–29.[Abstract/Free Full Text]
  12. Zeller KI, Jegga AG, Aronow BJ, O'Donnell KA, Dang CV. An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets. Genome Biol 2003;4:R69.[CrossRef][Medline]
  13. Li CM, Guo M, Borczuk A, et al. Gene expression in Wilms' tumor mimics the earliest committed stage in the metanephric mesenchymal-epithelial transition. Am J Pathol 2002;160:2181–90.[Abstract/Free Full Text]
  14. Wu KJ, Polack A, Dalla-Favera R. Coordinated regulation of iron-controlling genes, H-ferritin and IRP2, by c-MYC. Science 1999;283:676–9.[Abstract/Free Full Text]
  15. Wu KJ, Grandori C, Amacker M, et al. Direct activation of TERT transcription by c-MYC. Nat Genet 1999;21:220–4.[CrossRef][Medline]
  16. Cardiff RD, Sinn E, Muller W, Leder P. Transgenic oncogene mice. Tumor phenotype predicts genotype. Am J Pathol 1991;139:495–501.[Abstract]
  17. Wulf G, Garg P, Liou YC, Iglehart D, Lu KP. Modeling breast cancer in vivo and ex vivo reveals an essential role of Pin1 in tumorigenesis. EMBO J 2004;23:3397–407.[CrossRef][Medline]
  18. Aulmann S, Bentz M, Sinn HP. C-myc oncogene amplification in ductal carcinoma in situ of the breast. Breast Cancer Res Treat 2002;74:25–31.[CrossRef][Medline]
  19. Glockner S, Lehmann U, Wilke N, Kleeberger W, Langer F, Kreipe H. Amplification of growth regulatory genes in intraductal breast cancer is associated with higher nuclear grade but not with the progression to invasiveness. Lab Invest 2001;81:565–71.[Medline]
  20. Courjal F, Cuny M, Simony-Lafontaine J, et al. Mapping of DNA amplifications at 15 chromosomal localizations in 1875 breast tumors: definition of phenotypic groups. Cancer Res 1997;57:4360–7.[Abstract/Free Full Text]
  21. Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer 2004;4:143–53.[Medline]
  22. Haig D. Genomic imprinting and kinship: how good is the evidence? Annu Rev Genet 2004;38:553–85.[CrossRef][Medline]



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