J Breast Cancer. 2007 Mar;10(1):51-58. Korean.
Published online Mar 31, 2007.
Copyright © 2007 Korean Breast Cancer Society
Original Article

Methylation Patterns of Cancer-Associated Genes in Breast Cancer

Sung-Bae Jee, Woo-Chan Park, Kee-Whan Kim, Ji-Il Kim, Chang-Hyeok Ahn, Keun-Woo Lim, Se-Jung Oh, Byung-Joo Song, Sang-Seol Jung and Jeong-Soo Kim
    • Department of Surgery, The Catholic University of Korea, College of Medicine, Seoul, Korea.
Received December 11, 2006; Accepted February 23, 2007.

Abstract

Purpose

To investigate the methylation status of cancer-associated genes in breast cancer to assess its use in the diagnosis of breast cancer and the relationship with distinctive clinical and pathological features.

Methods

A total of 29 benign tumors and their adjacent normal tissues as well as 67 malignant tumors and adjacent normal samples, from women undergoing surgery for primary invasive breast carcinoma at Uijongbu St. Mary's Hospital, between March 2003 and March 2005, were used. Eleven candidate genes were chosen; P14, P16, DAPK, MGMT, h-MLH, E-cadherin, RASSF1α, Twist, RARβ, HIN-1, and Cyclin D. DNA was extracted from fresh tissues, and methylation specific PCR performed.

Results

The number of methylated genes was increased in the malignant tissues compared to the benign tumors and adjacent normal tissues. 7 genes; P14, P16, MGMT, RASSF1α, Twist, RARβ, and Cyclin D, were more frequently methylated in malignant than benign tumors, with the differences in the p14, p16, and RARβ, genes were statistically significant (p<0.05). In benign tumors, the p16 and HIN-1 genes were the most infrequently (6.9%) and frequently methylated (82.8%), respectively. In malignant tumors, the h-MLH and RASSF1α genes were most infrequently and frequently methylated genes, respectively. The subgroup showing methylation of the DAPK gene had a higher nuclear grade and greater progesterone receptor negativity. The group in which the RASSF1α gene was methylated, had greater estrogen receptor (ER) and progesterone receptor (PgR) positivities. The Twist gene was frequently methylated in the subgroup showing higher nuclear and histologic grades. The group with HIN-1 and cyclin D methylation had a tendency to show greater ER positivity.

Conclusion

The subgroups showing methylated DAPK and Twist should be more intensely treated and followed up more carefully than those with RASSF1α, HIN-1 and Cyclin D methylation. Gene methylation may be linked to various pathological features of breast cancer; however, this will require confirmation from larger studies.

Keywords
Breast cancer; Tumor suppressor gene; Methylation

Figures

Fig 1
Number of methylated genes, benign tumor vs. normal tissue.

Fig 2
Number of methylated genes, malignant tumor vs. normal tissue.

Fig 3
The correlation of numbers of methylated genes between normal tissue vs. malignant tumor.

Fig 4
The correlation of numbers of methylated genes between normal tissue vs. benign tumor.

Tables

Table 1
Genes investigated in this study

Table 2
Characteristics of breast cancer patients

Table 3
MS-PCR primers of specific genes analyzed in this study

Table 4
Comparision of numbers of hypermethylation in 11 genes

Table 5
The comparison of methylation status of each gene, benign tumor vs. malignant tumor

Table 6
The comparison of methylation status of each gene, normal tissue vs. malignant tumor

Table 7
Logistic regression of hypermethylation status, benign tumor vs. malignant tumor

Table 8
Logistic regression of hypermethylation status, normal tissue vs. malignant tumor

Table 9
Association between gene promoter methylation and clinicopathological features

References

    1. El-Osta A. The rise and fall of genomic methylation in cancer. Leukemia 2004;18:233–237.
    1. Garinis GA, Patrinos GP, Spanakis NE, Menounos PG. DNA hypermethylation: when tumour suppressor genes go silent. Hum Genet 2002;111:115–127.
    1. Szyf M, Pakneshan P, Rabbani SA. DNA methylation and breast cancer. Biochem Pharmacol 2004;68:1187–1197.
    1. Widschwendter M, Jones PA. DNA methylation and breast carcinogenesis. Oncogene 2002;21:5462–5482.
    1. Esteller M. Dormant hypermethylated tumour suppressor genes: questions and answers. J Pathol 2005;205:172–180.
    1. van Rijnsoever M, Grieu F, Elsaleh H, Joseph D, Iacopetta B. Characterisation of colorectal cancers showing hypermethylation at multiple CpG islands. Gut 2002;51:797–802.
    1. Yan PS, Perry MR, Laux DE, Asare AL, Caldwell CW, Huang TH. CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer. Clin Cancer Res 2000;6:1432–1438.
    1. Fackler MJ, McVeigh M, Evron E, Garrett E, Mehrotra J, Polyak K, et al. DNA methylation of RASSF1α, HIN-1, RAR-beta, Cyclin D2 and Twist in in situ and invasive lobular breast carcinoma. Int J Cancer 2003;107:970–975.
    1. Lehmann U, Langer F, Feist H, Glockner S, Hasemeier B, Kreipe H. Quantitative assessment of promoter hypermethylation during breast cancer development. Am J Pathol 2002;160:605–612.
    1. Garcia JM, Silva J, Pena C, Garcia V, Rodriguez R, Cruz MA, et al. Promoter methylation of the PTEN gene is a common molecular change in breast cancer. Genes Chromosomes Cancer 2004;41:117–124.
    1. Bae YK, Brown A, Garrett E, Bornman D, Fackler MJ, Sukumar S, et al. Hypermethylation in histologically distinct classes of breast cancer. Clin Cancer Res 2004;10:5998–6005.
    1. Mason SL, Loughran O. La Thangue NB. P14(ARF) regulates E2F activity. Oncogene 2002;21:4220–4230.
    1. Enders GH, Koh J, Missero C, Rustgi AK, Harlow E. P16 inhibition of transformed and primary squamous epithelial cells. Oncogene 1996;12:1239–1245.
    1. Liu Y, Lee MO, Wang HG, Li Y, Hashimoto Y, Klaus M, et al. Retinoic acid receptor beta mediates the growth-inhibitory effect of retinoic acid by promoting apoptosis in human breast cancer cells. Mol Cell Biol 1996;16:1138–1149.
    1. Sabharwal A, Middleton MR. Exploiting the role of O(6)-methylguanine-DNA-methyltransferase (MGMT) in cancer therapy. Curr Opin Pharmacol 2006;6:355–363.
    1. Burbee DG, Forgacs E, Zochbauer-Muller S, Shivakumar L, Fong K, Gao B, et al. Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst 2001;93:691–699.
    1. Shapiro GI. Cyclin-dependent kinase pathways as targets for cancer treatment. J Clin Oncol 2006;24:1770–1783.
    1. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 2004;117:927–939.
    1. Cohen O, Feinstein E, Kimchi A. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J 1997;16:998–1008.
    1. Shigematsu H, Suzuki M, Takahashi T, Miyajima K, Toyooka S, Shivapurkar N, et al. Aberrant methylation of HIN-1 (high in normal-1) is a frequent event in many human malignancies. Int J Cancer 2005;113:600–604.
    1. Soreide K, Janssen EA, Soiland H, Korner H, Baak JP. Microsatellite instability in colorectal cancer. Br J Surg 2006;93:395–406.
    1. Asgeirsson KS, Jónasson JG, Tryggvadóttir L, Olafsdóttir K, Sigurgeirsdóttir JR, Ingvarsson S, et al. Altered expression of E-cadherin in breast cancer. Patterns, mechanisms and clinical significance. Eur J Cancer 2000;36:1098–1106.
    1. Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP. CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA 1999;96:8681–8686.
    1. Ward RL, Cheong K, Ku SL, Meagher A, O'Connor T, Hawkins NJ. Adverse prognostic effect of methylation in colorectal cancer is reversed by microsatellite instability. J Clin Oncol 2003;21:3729–3736.
    1. Widschwendter M, Siegmund KD, Muller HM, Fiegl H, Marth C, Muller-Holzner E, et al. Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen. Cancer Res 2004;64:3807–3813.

Metrics
Share
Figures

1 / 4

Tables

1 / 9

PERMALINK