Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Tumor Immunology: New Perspectives
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 5449-5451, November 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pedersen, I. S.
Right arrow Articles by McCann, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pedersen, I. S.
Right arrow Articles by McCann, A.
[Cancer Research 59, 5449-5451, November 1, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Frequent Loss of Imprinting of PEG1/MEST in Invasive Breast Cancer

Inge S. Pedersen, Peter A. Dervan, Dennise Broderick, Michèele Harrison, Nicola Miller, Emma Delany, Donal O’Shea, Paul Costello, Alo McGoldrick, George Keating, Brendan Tobin, Tom Gorey and Amanda McCann1

Department of Pathology, Biotechnology Centre, University College Dublin, Belfield, Dublin 4 [I. S. P., P. A. D., D. B., E. D., P. C., A. McG., A. McC.]; Departments of Histopathology [P. A. D., M. H., G. K., B. T.] and Surgery [T. G.], Mater Hospital, Dublin 7; National Centre for Medical Genetics, Crumlin, Dublin 12 [N. M.]; and Bio Research Ireland, Biotechnology Centre, University College Dublin, Belfield, Dublin 4 [D. O.], Ireland


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
The human PEG1 gene is a newly identified imprinted gene on 7q32. Genetic aberrations of this chromosomal region are often detected in invasive breast carcinomas. In this study, we show monoallelic PEG1 expression in normal breast tissue, indicating the presence of a functional imprint, and more importantly, we demonstrate loss of imprinting (LOI) in all of seven informative invasive breast carcinomas. In contrast to this, in one case of atypical ductal hyperplasia (ADH) found in residual breast, imprinting was maintained. This raises the possibility that aberrant imprinting of PEG1 may be involved in the progression from hyperplasia to invasive breast cancer.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
Genomic imprinting refers to the mechanism by which a number of genes throughout the genome are monoallelically expressed according to their parental origin. Epigenetic alteration in the form of reactivation of a normally silent/imprinted allele (LOI2 ) is a well-established mechanism for overexpression of the growth-promoting gene, IGF2, in cancer (1, 2, 3, 4, 5) . A detailed knowledge of the involvement of imprinted genes in cancer is of utmost importance, because aberrant imprinting appears far more accessible to therapeutic intervention than genetic changes. The in vitro treatment of tumor cells with 5-aza-2'-deoxycytidine, a specific inhibitor of cytosine DNA methyltransferase, has been reported to reverse LOI of IGF2 and H19 (6) .

In this study, we have investigated the allelic usage of the imprinted gene PEG1 in breast cancer. PEG1 is located on chromosome 7. Several lines of evidence suggest the involvement of genes on chromosome 7 in tumor progression and invasion. Cytogenetic studies show that trisomy 7 is one of the most frequent numerical chromosomal aberrations in breast tumor cell lines (7, 8, 9) . Structural changes of chromosome 7 also appear in breast cancer, one of the more frequent being the LOH of several markers in the 7q31–32 region. One group reported 40.5% LOH on 7q31 and suggested that this might be the site of a breast tumor or metastasis suppressor gene (10) . More recently, an international collaborative study group (The Breast Cancer Somatic Genetics Consortium) reported on their combined results on 683 breast tumors. LOH scorings for three polymorphic markers on 7q31–32 gave an average rate of 19% (11) . The only imprinted gene identified on chromosome 7 thus far is PEG1, located at 7q32 (12) . The biological function of PEG1 is unknown. However, the putative protein shares amino acid homology with the {alpha}/{beta} hydrolase fold family, which also includes the lysosomal enzyme cathepsin A. This could suggest a possible role in degradation of the extracellular matrix in the invasive state of tumor development. Peg1/Mest knockout mice show abnormal maternal behavior and growth retardation and have smaller placentae (13) . This indicates that Peg1 is either directly involved in the regulation of growth of the fetus and/or placenta.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Tissue Samples.
Frozen breast tumor samples and adjacent normal breast tissue were prospectively collected from the Mater Misericordiae Hospital (Dublin, Ireland). Tissue samples from a total of 53 patients were analyzed. The sample cohort consisted of 20 infiltrating carcinomas (16 ductal, 1 lobular, and 3 miscellaneous), 3 pure DCIS, 19 mixtures of both invasive and in situ components, and 5 benign lesions (2 fibrocystic changes, 2 phylloides tumors, and 1 myoepithelioma). In addition to these 47 primary lesions, 6 residual breast samples were analyzed. DCIS was found in one sample, and the remaining five were benign lesions (three fibrocystic changes, one combined fibroadenoma and fibrocystic change, and one ADH).

Histology.
All invasive carcinomas were graded using a modified Bloom and Richardson grading system (14) , and the DCIS were graded by nuclear morphology (15) . The proportion of the tumor occupied by a DCIS component was assessed visually. The lymphocytic response to the tumor was assessed using a grading system as follows: mild, a small number of lymphocytes scattered around tumor cells; severe, an intense lymphocytic infiltrate as might be seen in medullary carcinoma; and moderate, midway between the two. The infiltrate was said to be focal if it occupied <10% of the tumor area and was confined to one or two areas.

PCR Amplification of Genomic DNA and RT-PCR.
Primers were designed to encompass the AflIII RFLP in the 3' untranslated region of the PEG1 cDNA (GenBank accession no. D78611). High molecular weight DNA was extracted from snap frozen tissue using standard methods. PCR reactions were set up on 0.5 µg of genomic DNA using primers PegF (5'-TAC TAA ACC AGC ATA CCC TTA C-3') and PegR (5'-GCA GTC ATC ATA AAG GAA TCA G-3') in 50-µl reactions containing 1x Taq buffer, 250 µM deoxynucleotide triphosphate mixture, 80 pmol of each primer, and 1.25 unit of Taq polymerase (Promega). The reactants were subjected to 5 min of initial denaturing at 95°C, followed by 35 cycles (95°C for 30 s, 50°C for 30 s, and 72°C for 30 s) with a final extension for 7 min at 72°C. PCR was carried out on a DNA Engine (PTC-2000).

RNA from informative patients was extracted using the TRIzol RNA extraction kit (Life Technologies, Inc.), according to manufacturer’s instructions. To eliminate any contaminating genomic DNA, 1 µg of RNA was DNase treated using DNaseI (Life Technologies, Inc.), and 0.5 µg was used as a template in the Access RT-PCR kit (Promega) using primers PegF and PegR. The remaining 0.5-µg aliquot of DNase-treated RNA was set up in the absence of the AMV enzyme and hence served as a control for DNA contamination. Cycling conditions were as follows: 48°C for 45 min, followed by 95°C for 5 min, and 40 cycles of 95°C for 30 s, 50°C for 1 min, and 68°C for 2 min, and finally, 7 min at 68°C.

Determination of Heterozygosity and Allelic Usage.
AflIII restriction digestion of 10 µl of PCR or RT-PCR product was carried out in 15-µl reactions containing 1 unit of AflIII enzyme and 1x REact3 buffer (Life Technologies, Inc.) for 4 h at 37°C.

Image Analysis.
Digital images of gels were captured in 8-bit grayscale (256 gray values) and were processed in the following manner. The image grayscale values were inverted, resulting in an image with dark bands on a light background. The resultant image was normalized by stretching the histogram, setting the median grayscale value of the image (background) to 255 (white), and adjusting all other values upward on a factorial basis. This resulted in an image with dark bands on white background. Bands of interest were selected, and the following parameter was calculated:

where P is the integrated log inverse grayscale value (absorbance) for the band, N is the number of pixels in the area, and Ii is the intensity of pixel i. Ratios of bands were calculated by comparing the calculated P of the less intense band to the P of the more intense band.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
The PEG1-specific PCR product was amplifiable from genomic DNA of 53 paired normal and tumor samples. Of these 53 patients, 12 (23%) were informative for the AflIII polymorphism (9 invasive carcinomas, 1 DCIS, 1 fibrocystic change, and 1 ADH in residual breast). All but one sample had retained heterozygosity in the abnormal tissue (Fig. 1)Citation . The one sample displaying LOH was a high-grade DCIS, comedo type. RNA analysis of this sample established that the lost allele was the inactive copy (Fig. 2)Citation . PEG1 RT-PCR products from 8 of the 11 samples with retained heterozygosity were analyzable by restriction digestion. Seven tumors, all invasive, showed biallelic expression (LOI), whereas a functional imprint, evident by monoallelic expression, was in place in the paired normal samples (Fig. 2)Citation . One sample had maintained imprinting in the abnormal tissue. This was a case of ADH.



View larger version (47K):
[in this window]
[in a new window]
  		Fig. 1. Heterozygosity of the AflIII polymorphism. AflIII restriction digests of paired normal (N) and tumor (T) samples run side by side. Samples 96 and 88 are examples of uninformative patients. Sample 98 is heterozygous with retained heterozygosity in the tumor (T98). Sample 89 is also heterozygous but shows LOH in the tumor (T89).

 


View larger version (43K):
[in this window]
[in a new window]
  		Fig. 2. Imprinting status of paired normal and tumor samples. All normal samples (N) have an imprinted phenotype, whereas there is LOI in the tumor tissue of sample 39, 4, and 53 (T39, T4, and T53). T56 has maintained imprinting in the abnormal tissue. Sample 89 is the sample with LOH of the lower allele (Fig. 1)Citation . This gel shows that the retained allele is the active copy. The DNA marker (M) used as size standard is {phi}174 DNA/HaeIII (Promega).

 
LOI was measured semiquantitatively by comparing the absorbance of the gel bands representing the two alleles. All but one of the cases showing reactivation of the silent allele had ratios of the less abundant to the more abundant allele of more than 1:2 (Table 1)Citation . These cases would be classified as LOI using the arbitrarily chosen threshold of 1:3 applied by Cui et al. (16) for IGF2. One sample, however, had a ratio of 1:4, which could be a result of contaminating normal tissue or maintained imprinting in the coexisting DCIS component (DCIS accounts for 30% of the tumor mass in this case). Of note is the observation that three samples with allelic ratios approaching or reaching 1:1 were the only tumors without a DCIS component. This indicates the possibility of a normal imprinted phenotype in the noninvasive DCIS tumors, which would further support the theory that LOI of PEG1 is involved in tumor invasion. In agreement with previous reports, we found that the repression of PEG1 is incomplete (12) , with transcription levels of the silent allele reaching 1:20 to 1:10 of the expressed allele.


View this table:
[in this window]
[in a new window]
  		Table 1 Histological details of tumors Case

 
PEG1 is biallelically expressed in adult lymphocytes (17) . To ensure that the LOI observed in the tumors is not an artifact resulting from a lymphocytic response to the tumor, all tissue samples were stained with H&E, and an assessment was made of the number of lymphocytes present (Table 1)Citation . The degree of lymphocytic infiltrate present in the tumors appears unlikely to account for the biallelic profiles.

The consistent finding of PEG1 LOI in invasive breast carcinomas suggests a possible role for this imprinted gene in breast carcinoma progression and/or invasion. We were surprised by this finding, because of earlier reports of LOH in the 7q31–32 region, suggesting the existence of a tumor suppressor gene at this site. Because LOH in many cases is accompanied by duplication of the retained allele (as a result of gene conversion or mitotic recombination), it is possible that the outcome of LOH at an imprinted locus equals the outcome of LOI, i.e., two active gene copies. Alternatively, the existence of a reciprocal imprinted gene in close proximity to PEG1, the imprinting status of which is coregulated in a manner similar to H19 and IGF2, would also account for the apparent involvement of both LOH and LOI of 7q31–32. Whether PEG1 is part of a larger imprinting domain remains to be determined.

Investigation of the mechanism of PEG1 LOI in breast cancer would be facilitated by methylation analysis of the PEG1 promoter. In fetal samples, the promoter of the inactive maternal allele is methylated, whereas the paternal promoter is unmethylated (17) . Interestingly, although this differential methylation is conserved in adult lymphocytes, these cells display biallelic expression (17) . It would be of interest to establish whether the methylation of the promoter region is altered in cancer tissue with biallelic expression. This may suggest that different mechanisms underlie the normal tissue-specific biallelic expression of PEG1 and the biallelic expression observed in breast cancer.

PEG1 is also known as mesodermal-specific transcript (MEST) because of its preferential transcription in tissue of mesodermal origin. We did, however, detect PEG1 expression in the epithelial breast cancer cell lines MCF7 and MCF10 using Northern blot analysis (data not shown). The expression levels were ~4-fold higher in the invasive MCF7 cell line compared with the immortalized MCF10 cells. This further supports the possibility of PEG1 being one of the genes on chromosome 7 involved in the stages of local tumor invasion.


   ACKNOWLEDGMENTS
 
We are very grateful for the cell lines generously provided by Dr. David Easty, Department of Pathology, University College Dublin, Dublin, Ireland, and for his assistance with the Northern blots.


   FOOTNOTES
 
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.

1 To whom requests for reprints should be addressed, at Department of Pathology, Biotechnology Center, University College Dublin, Belfield, Dublin 4, Ireland. E-mail: amccann{at}ollamh.ucd.ie Back

2 The abbreviations used are: LOI, loss of imprinting; IGF, insulin-like growth factor; LOH, loss of heterozygosity; DCIS, ductal carcinoma in situ; ADH, atypical ductal hyperplasia; RT-PCR, reverse transcription-PCR. Back

Received 6/28/99. Accepted 8/19/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

  1. Rainier S., Johnson L. A., Dobry C. J., Ping A. J., Grundy P. E., Feinberg A. P. Relaxation of imprinted genes in human cancer. Nature (Lond.), 362: 747-749, 1993.[Medline]
  2. Ogawa O., McNoe L. A., Eccles M. R., Morison I. M., Reeve A. E. Relaxation of insulin-like growth factor II gene imprinting implicated in Wilms’ tumor. Nature (Lond.), 362: 749-751, 1993.[Medline]
  3. McCann A. H., Miller N., O’Meara A., Pedersen I., Keogh K., Gorey T., Dervan P. A. Biallelic expression of the IGF2 gene in human breast cancer. Hum. Mol. Genet., 5: 1123-1127, 1996.[Abstract/Free Full Text]
  4. Nonomura N., Nishimura K., Miki T., Kanno N, Kojima Y., Yokoyama M., Okuyama A. Loss of imprinting of the insulin-like growth factor II gene in renal cell carcinoma. Cancer Res., 57: 2575-2577, 1997.[Abstract/Free Full Text]
  5. Wu M. S., Wang H. P., Lin C. C., Sheu J. C., Lee W. J., Lin J. T. Loss of imprinting and overexpression of IGF2 in gastric adenocarcinoma. Cancer Lett., 120: 9-14, 1997.[Medline]
  6. Barletta J. M., Rainier S., Feinberg A. P. Reversal of loss of imprinting in tumor cells by 5-aza-2'-deoxycytidine. Cancer Res., 57: 48-50, 1997.[Abstract/Free Full Text]
  7. Gebhart E., Brüderlein S., Augustus M., Siebert E., Feldner J., Schmidt W. Cytogenetic studies on human breast carcinomas. Breast Cancer Res. Treat., 8: 125-138, 1986.[Medline]
  8. Rodgers C. S., Hill S. M., Hultén M. A. Cytogenetic analysis in human breast carcinoma. I. Nine cases in the diploid range investigated using direct preparations. Cancer Genet. Cytogenet., 13: 95-119, 1984.[Medline]
  9. Geleick D., Müller H., Matter A., Torhorst J., Regenass U. Cytogenetics of breast cancer. Cancer Genet. Cytogenet., 46: 217-229, 1990.[Medline]
  10. Bièeche I., Champèeme M. H., Matifas F., Hacèene K., Callahan R., Lidereau R. Loss of heterozygosity on chromosome 7q and aggressive primary breast cancer. Lancet, 339: 139-143, 1992.[Medline]
  11. Devilee P., Hermans J., Eyfjord J., Booresen A. L., Liderau R., Sobol H., Borg A., Cleton-Jansen A. M., Olah E., Cohen B. B., Scherneck S., Hamann U., Peterlin B., Caligo M., Bignon Y. J., Maugard C. Loss of heterozygosity at 7q31 in breast cancer: results from an International Collaborative Study Group. The Breast Cancer Somatic Genetics Consortium. Genes Chromosomes Cancer, 18: 193-199, 1997.[Medline]
  12. Kobayashi S., Kohda T., Miyoshi N., Kuroiwa Y., Aisaka K., Tsutsumi O., Kaneko-Ishino T., Ishino F. Human PEG1/MEST, an imprinted gene on chromosome 7. Hum. Mol. Genet., 6: 781-786, 1997.[Abstract/Free Full Text]
  13. Lefebvre L., Viville S., Barton S. C., Ishino F., Keverne E. B., Surani M. A. Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest.. Nature Genet., 20: 163-169, 1998.[Medline]
  14. Elston C. W., Ellis I. O. Pathological prognostic factors in breast cancer. 1. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology, 19: 403-410, 1991.[Medline]
  15. Lennington W. J., Jensen R. A., Dalton W. L., Page D. L. Ductal carcinoma in situ of the breast. Heterogeneity of individual lesions. Cancer (Phila.), 73: 118-124, 1994.[Medline]
  16. Cui H., Horon I. L., Ohlsson R., Hamilton S. R., Feinberg A. P. Loss of imprinting in normal tissue of colorectal cancer patients with microsatellite instability. Nat. Med., 4: 1276-1280, 1998.[Medline]
  17. Riesewijk A. M., Hu L., Schulz U., Tariveridan G., Höglund P., Kere J., Ropers H-H., Kalsheuer V. M. Monoallelic expression of human PEG1/MEST is paralleled by parent-specific methylation in fetuses. Genomics, 42: 236-244, 1997.[Medline]



This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
P. J. Rugg-Gunn, A. C. Ferguson-Smith, and R. A. Pedersen
Status of genomic imprinting in human embryonic stem cells as revealed by a large cohort of independently derived and maintained lines
Hum. Mol. Genet., October 15, 2007; 16(R2): R243 - R251.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
V. A. Heinzelmann-Schwarz, M. Gardiner-Garden, S. M. Henshall, J. Scurry, R. A. Scolyer, M. J. Davies, M. Heinzelmann, L. H. Kalish, A. Bali, J. G. Kench, et al.
Overexpression of the Cell Adhesion Molecules DDR1, Claudin 3, and Ep-CAM in Metaplastic Ovarian Epithelium and Ovarian Cancer
Clin. Cancer Res., July 1, 2004; 10(13): 4427 - 4436.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
K. Woodson, A. Flood, L. Green, J. A. Tangrea, J. Hanson, B. Cash, A. Schatzkin, and P. Schoenfeld
Loss of Insulin-Like Growth Factor-II Imprinting and the Presence of Screen-Detected Colorectal Adenomas in Women
J Natl Cancer Inst, March 3, 2004; 96(5): 407 - 410.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
J.-C. Goffard, L. Jin, H. Mircescu, P. Van Hummelen, C. Ledent, J.-E. Dumont, and B. Corvilain
Gene Expression Profile in Thyroid of Transgenic Mice Overexpressing the Adenosine Receptor 2a
Mol. Endocrinol., January 1, 2004; 18(1): 194 - 213.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Yuan, R. Z. Luo, S. Fujii, L. Wang, W. Hu, M. Andreeff, Y. Pan, M. Kadota, M. Oshimura, A. A. Sahin, et al.
Aberrant Methylation and Silencing of ARHI, an Imprinted Tumor Suppressor Gene in which the Function Is Lost in Breast Cancers
Cancer Res., July 15, 2003; 63(14): 4174 - 4180.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Cui, P. Onyango, S. Brandenburg, Y. Wu, C.-L. Hsieh, and A. P. Feinberg
Loss of Imprinting in Colorectal Cancer Linked to Hypomethylation of H19 and IGF2
Cancer Res., November 15, 2002; 62(22): 6442 - 6446.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
I. S. Pedersen, P. Dervan, A. McGoldrick, M. Harrison, F. Ponchel, V. Speirs, J. D. Isaacs, T. Gorey, and A. McCann
Promoter switch: a novel mechanism causing biallelic PEG1/MEST expression in invasive breast cancer
Hum. Mol. Genet., June 1, 2002; 11(12): 1449 - 1453.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Li, T. H. Vu, K.-O. Lee, Y. Yang, C. V. Nguyen, H. Q. Bui, Z.-L. Zeng, B. T. Nguyen, J.-F. Hu, S. K. Murphy, et al.
An Imprinted PEG1/MEST Antisense Expressed Predominantly in Human Testis and in Mature Spermatozoa
J. Biol. Chem., April 12, 2002; 277(16): 13518 - 13527.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pedersen, I. S.
Right arrow Articles by McCann, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pedersen, I. S.
Right arrow Articles by McCann, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online