Very High Frequency of Hypermethylated Genes in Breast Cancer Metastasis to the Bone, Brain, and Lung

Abstract

Purpose: Most often it is not the primary tumor, but metastasis to distant organs that results in the death of breast cancer patients. To characterize molecular alterations in breast cancer metastasis, we investigated the frequency of hypermethylation of five genes (Cyclin D2, RAR-␤, Twist, RASSF1A, and HIN-1) in metastasis to four common sites: lymph node, bone, brain, and lung.Experimental Design: Methylation-specific PCR for the five genes was performed on DNA extracted from archival paraffin-embedded specimens of paired primary breast cancer and its lymph nodes (LN) metastasis (n ‫؍‬ 25 each); in independent samples of metastasis to the bone (n ‫؍‬ 12), brain (n ‫؍‬ 8), and lung (n ‫؍‬ 10); and in normal bone, brain, and lung (n ‫؍‬ 22).Results: No hypermethylation was detected in the five genes in the normal host tissues. In paired samples, LN metastasis had a trend of higher prevalence of methylation compared with the primary breast carcinoma for all five genes with significance for HIN-1 (P ‫؍‬ 0.04). Compared with the primary breast carcinomas, all five genes had higher methylation frequencies in the bone, brain, and lung metastasis, with HIN-1 and RAR-␤ methylation being significantly higher (P < 0.01) in each group. Loss of expression of all five genes correlated, with a few exceptions, to hypermethylation of their promoter sequences in metastatic carcinoma cells microdissected from LNs.Conclusion: The frequent presence of hypermethylated genes in locoregional and distant metastasis could render them particularly susceptible to therapy targeted toward gene reactivation combining demethylating agents, histone deacetylase inhibitors, and/or differentiating agents.

DNA hypermethylation and clinicopathological features in breast cancer: the Western New York Exposures and Breast Cancer (WEB) Study

“…The gene is methylated frequently in breast cancer [14,15,18] and even normal breast tissue [38], which may result in loss of expression and a loss of control of cellular proliferation. Unlike previous studies, in which there was found a positive association between RAR-β 2 hypermethylation and metastasis in breast tumor [15,18], we found no associations between hypermethylation of RAR-β 2 gene and metastasis and lymphovascular invasion. However, we found inverse associations between histological and nuclear grade and RAR-β 2 hypermethylation among postmenopausal women.…”

“…Some studies have evaluated the association between gene hypermethylation and biological or clinical properties of breast tumors [13][14][15][16][17][18][19]. An association between CpG island hypermethylation of p16 and RAR-β 2 genes and poorly differentiated breast tumors was found in two previous studies [14,19].…”

“…Promoter hypermethylation of PTEN (phosphatase and tensin homologue deleted on chromosome 10) was associated with larger tumor size and higher histologic grade [17]. Methylation of GSTP1 (glutathione S-transferase π 1) and/or RAR-β 2 was found to be associated with breast cancer cases with sentinel lymph node metastasis [15]; and HIN-1 (high in normal-1) and RAR-β 2 had greatly higher methylation frequencies in bone, brain, and lung metastases than the primary breast tumors [18]. Some studies reported no apparent association between methylation distribution phenotypes and tumor size, grade, stage, or lymph node status [13,16].…”

Breast Cancer Metastasis to the Central Nervous System

“…47,48 No significant differences in the expression levels of p53, Bcl-2, E-cadherin, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, epidermal growth factor (EGF) receptor, cyclooxygenase 2, or Bax were observed between the primary tumors and brain metastases. In contrast, Mehrotra and colleagues 49 identified a DNA hypermethylation phenotype in tissue blocks containing metastatic lesions of breast cancer, including eight brain metastatic lesions. Hypermethylation of cyclin D2, retinoic acid receptor-␤, and hin-1 occurred more frequently in brain metastases than in an unmatched cohort of primary breast carcinomas whereas hypermethylation of other genes, including twist and RassF1A, was not significantly different.…”

Loss of a Negative Feedback Loop Involving Pea3 and Cyclin D2 Is Required for Pea3-Induced Migration in Transformed Mammary Epithelial Cells

“…The mechanisms of cyclin d2 repression in breast tumors are poorly understood but are often correlated to a hypermethylation of its promoter region (23,45,46). The cyclin d2 gene is as well-frequently downregulated in various breast cancer cell lines and Cyclin D2 overexpression in mice mammary gland leads to inhibition of the Cyclin D1 oncogene phosphorylation and therefore activation (23,26,47).…”