Alu and LINE-1 Hypomethylation Is Associated with HER2 Enriched Subtype of Breast Cancer

Abstract

The changes in DNA methylation status in cancer cells are characterized by hypermethylation of promoter CpG islands and diffuse genomic hypomethylation. Alu and long interspersed nucleotide element-1 (LINE-1) are non-coding genomic repetitive sequences and methylation of these elements can be used as a surrogate marker for genome-wide methylation status. This study was designed to evaluate the changes of Alu and LINE-1 hypomethylation during breast cancer progression from normal to pre-invasive lesions and invasive breast cancer (IBC), and their relationship with characteristics of IBC. We analyzed the methylation status of Alu and LINE-1 in 145 cases of breast samples including normal breast tissue, atypical ductal hyperplasia/flat epithelial atypia (ADH/FEA), ductal carcinoma in situ (DCIS) and IBC, and another set of 129 cases of IBC by pyrosequencing. Alu methylation showed no significant changes during multistep progression of breast cancer, although it tended to decrease during the transition from DCIS to IBC. In contrast, LINE-1 methylation significantly decreased from normal to ADH/FEA, while it was similar in ADH/FEA, DCIS and IBC. In IBC, Alu hypomethylation correlated with negative estrogen receptor (ER) status, and LINE-1 hypomethylation was associated with negative ER status, ERBB2 (HER2) amplification and p53 overexpression. Alu and LINE-1 methylation status was significantly different between breast cancer subtypes, and the HER2 enriched subtype had lowest methylation levels. In survival analyses, low Alu methylation status tended to be associated with poor disease-free survival of the patients. Our findings suggest that LINE-1 hypomethylation is an early event and Alu hypomethylation is probably a late event during breast cancer progression, and prominent hypomethylation of Alu and LINE-1 in HER2 enriched subtype may be related to chromosomal instability of this specific subtype.

Associations between genetic variation in one-carbon metabolism and LINE-1 DNA methylation in histologically normal breast tissues

“…Individuals undergo extensive changes in DNA methylation patterns during their lifespan, in accordance with the changes in their tissue-specific gene expression patterns related to aging and other physiologic states. 37 It is well established that alterations in DNA methylation represent early events in carcinogenesis, [1][2][3][4][5][6][7][8]24 characterized by genome-wide hypomethylation, especially at repetitive elements throughout the genome, such as LINE-1, and gene-specific promoter hypermethylation, which affects expression of tumor suppressor genes. 1-6, 25,35, 38 Histologically normal tissues are known to maintain a high percent methylation of LINE-1 (ranging from approximately 7585%-).…”

“…Genome-wide DNA hypomethylation and gene promoter hypermethylation are early events in the carcinogenic process [1][2][3][4][5] and have been linked to chromosomal instability, gene reactivation, activation of proto-oncogenes, and higher frequencies of mutation and recombination events, [6][7][8][9][10] all of which are characteristics of cancer genomes, including cancers of the breast. [11][12][13] Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable elements in the human genome.…”

“…[20][21][22][23][24] Additionally, compared to histologically normal or normal-appearing tissues (adjacent to breast tumors), tumor tissues consistently have lower levels of DNA methylation (i.e., increased hypomethylation throughout the genome), even early in breast carcinogenesis. 4,5,25 Furthermore, there are reports of significant associations between LINE-1 methylation and breast tumor clinicopathological features, 3 as well as breast cancer prognosis. 3,4 Current evidence regarding associations between variants in the folate-mediated one-carbon metabolism pathway and DNA methylation suggests that this relationship is a complex one, with evidence supporting a role of this pathway in breast cancer.…”

“…4,5,25 Furthermore, there are reports of significant associations between LINE-1 methylation and breast tumor clinicopathological features, 3 as well as breast cancer prognosis. 3,4 Current evidence regarding associations between variants in the folate-mediated one-carbon metabolism pathway and DNA methylation suggests that this relationship is a complex one, with evidence supporting a role of this pathway in breast cancer. 26,27 Disruption of this metabolic pathway, through alterations of the bioavailability of specific enzymes or variation in key genes, could lead to altered genome-wide or gene-specific methylation levels with differing consequences by genomic region.…”

“…Few studies 3,35,36 have examined LINE-1 methylation patterns in histologically normal breast tissues from healthy subjects, and it is unclear what factors and/or exposures are associated with breast tissue-specific LINE-1 methylation. Relevant risk factors that are known to be important in DNA methylation and one-carbon metabolism include folate concentrations and genetic variation in genes encoding the enzymes that play ratelimiting roles in the one-carbon metabolism pathway.…”