Hypomethylation of long interspersed nuclear element-1 (LINE-1) leads to activation of proto-oncogenes in human colorectal cancer metastasis

Abstract

Objective: Hypomethylation of LINE-1 elements has emerged as a distinguishing feature in human cancers. Limited evidence indicates that some LINE-1 elements encode an additional internal antisense promoter, and increased hypomethylation of this region may lead to inadvertent activation of evolutionarily methylation-silenced downstream genes. However, the significance of this fundamental epigenetic mechanism in colorectal cancer (CRC) has not been investigated previously.

Design: We analysed tissue specimens from 77 CRC patients with matched sets of normal colonic mucosa, primary CRC tissues (PC), and liver metastasis tissues (LM). LINE-1 methylation levels were determined by quantitative bisulfite pyrosequencing. MET, RAB3IP and CHRM3 protein expression was determined by western blotting and IHC. MET proto-oncogene transcription and 5-hydroxymethylcytosine (5-hmc) were evaluated by quantitative real-time-PCR.

Results: Global LINE-1 methylation levels in LM were significantly lower compared with the matched PC (PC=66.2% vs LM=63.8%; p<0.001). More importantly, we observed that specific LINE-1 sequences residing within the intronic regions of multiple proto-oncogenes, MET (p<0.001), RAB3IP (p=0.05) and CHRM3 (p=0.01), were significantly hypomethylated in LM tissues compared with corresponding matched PC. Furthermore, reduced methylation of specific LINE-1 elements within the MET gene inversely correlated with induction of MET expression in CRC metastases (R=-0.44; p<0.0001). Finally, increased 5-hmc content was associated with LINE-1 hypomethylation.

Conclusions: Our results provide novel evidence that hypomethylation of specific LINE-1 elements permits inadvertent activation of methylation-silenced MET, RAB3IP and CHRM3 proto-oncogenes in CRC metastasis. Moreover, since 5-hmc content inversely correlated with LINE-1 hypomethylation in neoplastic tissues, our results provide important mechanistic insights into the fundamental processes underlying global DNA hypomethylation in human CRC.

Keywords: Colorectal Neoplasia; Methylation; Oncogenes.

Figure 1

Methylation status of global LINE-1 repeat elements correlates with colorectal cancer (CRC) carcinogenesis as well as liver metastasis. (A) Methylation status of global LINE-1 repeat elements in microsatellite instable (HCT116, RKO and SW48) and microsatellite stable (Caco-2, SW480 and SW620) CRC cell lines (*p<0.05, t test). (B) Methylation status of global LINE-1 element in 77 paired normal colonic mucosa (NM), primary CRC tissues (PC), and matched corresponding liver metastasis tissues (LM). (black cross marks represent mean methylation levels; grey vertical boxes in the pyrograms illustrate individual CpG sites analysed; ***p<0.001, t test).

Figure 2

Expression of MET is primarily regulated by hypomethylation of the LINE-1 elements within the host MET gene. (A) Schematic illustration of the local LINE-1 sequence in the MET gene (the red box represents the local LINE-1 element; black boxes represent exons; the red arrow indicates the LINE-1 antisense promoter; blue arrows represent pyrosequencing primers; orange bar indicates CpG island; green arrows represent qRT-PCR primers). (B) Methylation status and transcription status of L1-MET in colorectal cancer (CRC) cell lines was determined by quantitative pyrosequencing analysis and qRT-PCR, respectively. (C) Protein expression (western blotting) of host MET in CRC cell lines.

Figure 3

Hypomethylation of L1-MET induces transcription of the MET proto-oncogene in colorectal cancer (CRC) metastasis development. (A) Methylation status of L1-MET in normal colonic mucosa (NM), primary CRC tissues (PC), and matched corresponding liver metastasis tissues (LM) (black cross marks represent mean methylation levels; grey vertical boxes in the pyrograms illustrate individual CpG sites analysed; ***p<0.001, t test) (B) Expression status of the L1-MET transcript in PC and matched LM. (**p<0.01, t test) (C) Correlation analysis between L1-MET methylation levels (%) and expression status of L1-MET transcripts (Pearson’s correlation coefficient, R; p values, p; red line, linear regression line).

Figure 4

MET protein is highly expressed in liver metastasis (LM) compared with primary colorectal cancer (CRC) (PC). (A) Representative photomicrographs from a CRC with positive and negative MET protein expression. Among the 72 paired PC and LM tissues, (B) 22 cases showed overexpression of MET in LM, (C) 44 cases revealed comparable expression in PC and LM and (D) 6 cases displayed low expression in LM compared with matched PC.

Figure 5

The presence of 5-hydroxymethylcytosine (5-hmc) is related to demethylation of LINE-1 sequence in colorectal cancer (CRC) metastasis. (A) Glucosyltransferase catalyses the transfer of a glucose moiety from uridine diphosphoglucose onto the 5-hmc. (the red box indicates the transferred glucose moiety) (B) The quantity of 5-hmc at a CpG locus in global LINE-1 and (C) in local L1-MET in normal colonic mucosa (NM), primary CRC tissues (PC), and matched liver metastasis tissues (LM). (the blue bold ‘C’ character indicates the CpG locus analysed; *p<0.05, **p<0.01, ***p<0.001, t test).

Figure 6

A consolidated model for the activation mechanism of the MET proto-oncogene by LINE-1 hypomethylation in colorectal cancer (CRC) metastasis. In this model, hypomethylation of CpG sites is characterised by enhanced conversion of 5-mc (5-methylcytosine) to 5-c (5-cytosine), with an intermediary generation of 5-hmc (5-hydroylated methylcytosine). Enhanced CpG demethylation with consequent accumulation of 5-hmc prevents the binding of various DNA methyltransferases (DNMTs) and methyl binding proteins (MBPs) to CpG sites, triggering a cascade of events that favour DNA hypomethylation. Occurrence of DNA hypomethylation events in the context of LINE-1 elements can potentially permit activation of certain oncogenes, including the MET proto-oncogene which may result in accelerated CRC metastasis.