Hypomethylation of retrotransposable elements correlates with genomic instability in non‐small cell lung cancer

Abstract

LINE-1 and Alu elements are non-LTR retrotransposons, constituting together over 30% of the human genome and they are frequently hypomethylated in human tumors. A relationship between global hypomethylation and genomic instability has been shown, however, there is little evidence to suggest active role for hypomethylation-mediated reactivation of retroelements in human cancer. In our study, we examined by Pyrosequencing the methylation levels of LINE-1 and Alu sequences in 48 primary nonsmall cell carcinomas and their paired adjacent tissues. We demonstrate a significant reduction of the methylation levels of both elements (p 5 7.7 3 10 214 and 9.6 3 10 27 , respectively). The methylation indices of the 2 elements correlated (p 5 0.006), suggesting a possible common mechanism for their methylation maintenance. Genomic instability was measured utilizing 11 fluorescent microsatellite markers located on lung cancer hot-spot regions such as 3p, 5q 9p, 13q and 17p. Hypomethylation of both transposable elements was associated with increased genomic instability (LINE, p 5 7.1 3 10 25 ; Alu, p 5 0.008). The reduction of the methylation index of LINE-1 and Alu following treatment of 3 lung cell lines with 5-aza-2 0 -deoxycitidine, consistently resulted in increased expression of both elements. Our study demonstrates the strong link between hypomethylation of transposable elements with genomic instability in non-small cell lung cancer and provides early evidence for a potential active role of these elements in lung neoplasia. As demethylating agents are now entering lung cancer trials, it is imperative to gain a greater insight into the potential reactivation of silent retrotransposons in order to advance for the clinical utilization of epigenetics in cancer therapy. ' 2008 Wiley-Liss, Inc.Key words: LINE-1; Alu; hypomethylation; genomic instability; lung cancer LINE-1 elements are autonomous non-LTR retrotransposons comprising almost 21% of the human genome. 1 Although most of these elements are inactive, mostly due to 5 0 truncations, there are a number of 80-100 intact LINE-1 elements that remain capable of retrotransposition. 2,3 The full length human LINE-1 retrotransposon is about 6 kb consisting of a 5 0 UTR, two open reading frames (ORF 1 and ORF2), and a 3 0 UTR with a functional polyadenylation signal and a variable length poly-A tail. 1 The bicistronic mRNA of LINE-1 is generated by an internal promoter at the 5 0 UTR region. ORF1 encodes for p40, a 40 kDa RNA-binding protein with cis preference for LINE-1 RNA, while ORF2 encodes for a 150 kDa protein with 3 conserved domains. 4 LINE-1 retrotransposition is undertaken with the help of both ORF1 and ORF2 proteins through a target-primed reverse transcription mechanism (TPRT). 2 In normal human cells, transcription and retrotransposition of LINE-1 is suppressed by a variety of control mechanisms including methylation, siRNAs and transcription regulators. [4][5][6] As a result, cells manage to protect themselves from a number of adverse effects exerted by LINE-1 ...

TPL2 kinase is a suppressor of lung carcinogenesis

“…4E). Hypomethylation of LINE-1 serves as a surrogate marker of global methylation status (27), correlates with genomic instability (28), and has been identified as an independent marker of poor prognosis in early stage NSCLC (41). It is, thus, tempting to speculate that the down-regulation of TPL2 caused by miR-370 hypomethylation may impact on the early stages of the disease in humans, similar to the effects of TPL2 ablation on urethaneinduced lung cancer initiation in the mouse.…”

“…4D). Moreover, this reduction closely correlated with the methylation status of the retrotransposable long interspersed nuclear element-1 (LINE-1), a surrogate marker of global methylation (27,28), suggesting that miR-370 hypomethylation in NSCLC is most likely the result of global genome hypomethylation (Fig. 4E).…”

Type 2 diabetes mellitus in relation to global LINE-1 DNA methylation in peripheral blood: A cohort study

“…For the methylation analysis of the LINE-1 element we used the primer sequences previously published by Daskalos et al, 36 including 6 CpG sites. Supplementary Table 1 shows the primer sequences for the assay.…”

A decade of DNA methylation profiling in cancer: What have we learned?

“…Numerous other studies have focused on repetitive DNA elements as targets of hypomethylation in the cancer genome (Dante 1988, Costa et al 2006, Ehrlich et al 2006. Hypomethylation of repetitive elements has been associated with chromosomal instability in tumors, perhaps by facilitating aberrant recombination events (Richards et al 2009, Daskalos et al 2009, Nishida et al 2013). …”