In this study, we examined whether clinical, pathologic and molecular variables could potentially explain the wide population variability of LINE-1 methylation in colorectal cancer. Global DNA hypomethylation has been associated with genomic instability, and implicated in the development of various human cancers [3
]. To estimate global DNA methylation level, we measured tumor LINE-1 methylation, which has been correlated well with cellular 5-methylcytosine level (i.e., global DNA methylation level) in cancer tissues [10
]. This is not surprising because LINE-1 retrotransposon constitutes a substantial portion (~17%) of the entire human genome [9
]. LINE-1 methylation in colorectal cancer is highly variable [18
], and is strongly associated with survival among colon cancer patients [20
]. Therefore, accurate prediction of tumoral LINE-1 methylation level from clinical and pathological features, if possible, may be clinically useful. Furthermore, recent evidence suggests a non-stochastic component of variable LINE-1 methylation levels in synchronous colorectal cancers (i.e., two or more primary tumors in one individual) [21
]. Thus, it is of particular interest to identify clinical, environmental or tumoral factors, if any, which influence LINE-1 methylation in colorectal cancer [53
Although accumulating evidence has suggested global DNA hypomethylation in human cancers, the mechanisms eliciting this alteration are still unknown. Possible mechanisms include inadequate expression or regulation of DNA methyltransferases, insufficient levels of methyl group donors (i.e., disorder of one-carbon metabolism pathway), aberrant activation of DNA demethylases, and altered expression of chromatin regulators directing DNA methyltransferases [54
]. The mechanism by which global DNA hypomethylation may confer a poor prognosis (i.e., tumor progression) also remains speculative. Genome-wide DNA hypomethylation has been associated with genomic instability [5
], which may confer poor prognosis. Transcriptional dysregulation might be another possible mechanism, and activation of proto-oncogenes, endogenous retroviruses, or transposable elements might affect tumor aggressiveness. A third possible mechanism involves inflammatory mediators and tumor hypoxia; the latter has been associated with genomic DNA hypomethylation [57
]. Activation of hypoxia-inducible factor HIF1A has been associated with poor prognosis in colon cancer [58
]. A better understanding of relationship between LINE-1 hypomethylation and clinical, pathologic, or molecular feature may shed lights on these biological mechanisms of LINE-1 hypomethylation in human cancer.
The efficiency and precision of bisulfite conversion is very crucial for quantitative assays based on sodium bisulfite treatment of genomic DNA. As the non-CpG cytosine in LINE-1 repetitive sequences has been documented to be rarely methylated [33
], we used the non-CpG cytosine as a built-in control for bisulfite conversion efficiency and confirmed successful bisulfite conversion by complete conversion of this cytosine (Figure , arrows
). In addition, we have previously assessed precision of bisulfite treatment and precision of subsequent Pyrosequencing assay to measure LINE-1 methylation in paraffin-embedded colon cancers [32
]. We assessed precision of bisulfite conversion by repeating bisulfite treatment. Basically, we performed bisulfite conversion on seven different DNA specimen aliquots from each of 10 different colorectal cancers. Bisulfite-to-bisulfite (between-bisulfite treatment) standard deviation ranged from 0.4-2.4 (median, 1.2), indicating good precision of bisulfite conversion in terms of LINE-1 methylation measurement.
We have discovered the relationship between LINE-1 extreme hypomethylation (< 40) and younger age of onset (< 60 year old). The LINE-1 extreme hypomethylators appear to be separate from the normal distribution of LINE-1 methylation observed in the remaining majority of colorectal cancers. This group of colorectal cancers has not been described previously, because it is necessary to analyze a large number of molecularly and clinically-annotated colorectal cancers with precise LINE-1 methylation data. The relationship of LINE-1 extreme hypomethylation with earlier age of onset and poor prognosis [20
] support the presence of a distinct subtype of colorectal cancers with a unique pathogenic mechanism. Nonetheless, additional studies are necessary to confirm our findings. We are currently investigating whether this class of LINE-1 extreme hypomethylators may indeed be driven by different underlying molecular mechanisms than those inducing the normal distribution of LINE-1 methylation seen in the majority of colorectal cancers.
We have also found that the linear regression model explains only 8.4% of variability of LINE-1 methylation, and the 92% of the variability still remained unexplained. In addition, we have shown that multivariate logistic regression models do not significantly predict binary LINE-1 methylation outcomes. Our results imply that LINE-1 methylation in colorectal cancer can vary greatly even after accounting for the clinical, pathologic and molecular features (examined in this study), despite the uniformity within individual patients in cases of synchronous colorectal cancers [21
]. Our data could point to several interesting potential hypotheses for underlying biological mechanisms; First, genomic methylation levels and tumor progression could be co-influenced by a variety of environmental factors (e.g., smoking, alcohol, or dietary pattern) for each patient. Second, a stochastic element in the underlying regulatory network could be improperly downregulated according to genomic DNA methylation status. Third, specific genes responsible for tumor behavior (e.g., tumor progression) might be aberrantly regulated in cases of extreme hypomethylation. We await further studies to elucidate the exact mechanisms of LINE-1 hypomethylation in colorectal cancers.
Examination of epigenetic and genetic aberrations is important in cancer research [59
]. We utilized a quantitative Pyrosequencing assay for LINE-1 methylation, which is robust and can accurately quantify LINE-1 methylation level [10
]. Pyrosequencing technology can detect subtle differences in average LINE-1 methylation levels among different colon cancer subtypes [e.g., microsatellite instability (MSI)-high vs. microsatellite stable (MSS)] [18
], emphasizing the importance of the use of an accurate and precise method to measure LINE-1 methylation. Our previous survival data indicates that LINE-1 measurement by Pyrosequencing is highly significantly associated with patient outcome [20
]. Considering the high reproducibility of LINE-1 Pyrosequencing [10
], the large variation in LINE-1 methylation levels likely reflects true heterogeneity in global methylation levels among individual tumors. Inter-tumoral biological heterogeneity reflecting the LINE-1 methylation variation is also supported by the highly significant association between LINE-1 methylation level and patient survival [20
Molecular classification of colorectal cancer based on MSI and the CpG island methylator phenotype (CIMP) status is increasingly important, because MSI and CIMP status reflect global genomic and epigenomic aberrations in tumor cells. Both CIMP-high and MSI-high are inversely associated with LINE-1 hypomethylation, suggesting that CIMP/MSI and genomic hypomethylation may represent different pathways to colorectal cancer. In addition, in non-MSI-high tumors, chromosomal instability (CIN) is correlated with LINE-1 hypomethylation, supporting the possible link between genome-wide hypomethylation and CIN. Further studies are necessary to examine the exact mechanism of how genomic hypomethylation, CIMP, MSI and CIN interact in colorectal cancer development and contribute to colorectal cancer progression.
In the multivariate linear and logistic regression models, we included key molecular events that have been well documented in colorectal cancer [65
]. Those molecular features include CIMP, MSI and TP53, all of which are related with LINE-1 or global DNA hypomethylation [4
]. In addition, our tumor database has enabled us to include other molecular variables (such as CDKN1A expression, CTNNB1 score, PTGS2 expression, FASN expression, KRAS
mutation and PIK3CA
mutation, all of which have been related with MSI or CIMP), as well as clinical and pathologic variables. Therefore, our multivariate linear and logistic regression analysis was quite comprehensive.
Although the multivariate linear regression model cannot explain a wide variation of tumoral LINE-1 methylation, the model can provide us with useful information on the independent relations of LINE-1 methylation with various clinical, pathologic and molecular features of colorectal cancer. The possible relationship of LINE-1 methylation with signet ring cell component (p = 0.0003) and rectal location (vs. colon; p = 0.0046) are intriguing. The mechanism of this relation remains to be investigated. In contrast, we found no significant difference in LINE-1 methylation between right colon and left colon, which is in agreement with data on LINE-1 methylation in normal colonic mucosa [52
]. The relation between family history of colorectal cancer and LINE-1 hypomethylation (p = 0.0089) may imply the presence of genetic component in global DNA hypomethylation. Of note, we have previously reported that certain SNPs in one-carbon metabolism genes are not associated with LINE-1 hypomethylation [69
]. Nonetheless, any of associations with p > 0.0021 in this current study could be a chance finding due to multiple hypothesis testing. Additional independent studies are necessary to confirm these associations.