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Battaglin et al. J Cancer Metastasis Treat 2018;4:12 I http://dx.doi.org/10.20517/2394-4722.2018.04 Page 13 of 25

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epigenetic silencing through promoter DNA hypermethylation, diploid copy number and absence of TP53 .
CIMP status has been proposed as a promising prognostic marker for CRCs, however, several studies reported
contradictory results, possibly due to the overlap between the CIMP+ phenotype and the MSI-H phenotype,
associated in 30%-50% of cases with BRAF mutation . The lack of global consensus in defining CIMP+
[166]
tumors, together with these controversial results, has hindered the uptake of CIMP as a relevant biomarker in
clinical practice and further studies are warranted to explore its predictive and prognostic value .
[167]
Long interspersed nucleotide element-1 (LINE-1) methylation measured by pyrosequencing has been shown
[168]
to correlate with global DNA methylation levels . LINE-1 is a retrotransposon related to key CRC features
involved in the carcinogenesis process: LINE1 hypomethylation is associated with 18q loss of heterozygosity
(LOH); whereas an inverse correlation has been demonstrated between LINE-1 hypomethylation,
CIMP-H and MSI-H status. LINE-1 methylation levels have been reported to impact CRC prognosis with
hypomethylation conferring poor prognosis in terms of overall mortality (OM) and colorectal cancer-
specific mortality . Additionally, LINE-1 hypomethylation in MSS/CIMP+ stage II and III CRC has been
[169]
[170]
showed to predict benefit from adjuvant chemotherapy with oral fluoropyrimidines . These data suggest
that DNA demethylation may play, as well, a crucial role in CRC development, prognosis and response to
treatment. Although promising, however, these findings need further validation.

The DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) has recently gained attention and
has been object of several studies. This gene encodes a DNA repair protein which removes alkylating groups
from O6-guanine and is involved in protecting cells against damages from alkylating agents. MGMT has
been shown to undergo epigenetic silencing by promoter hypermethylation in more than 40% of mCRCs .
[171]
The loss of MGMT gene expression impairs the ability of DNA repair mechanisms to remove alkyl groups,
potentially enhancing the cytotoxic effects of alkylating drugs, such as dacarbazine and temozolomide.
[172]
On these bases, several phase II clinical trials evaluating the efficacy of alkylating agents in mCRC have
been conducted with promising results. In these studies, MGMT methylation has been used as a predictive
biomarker for patients’ selection, supporting a possible role for this novel marker in clinical practice.

In an era in which immuno-oncology is revolutionizing cancer treatment strategies, novel possible relevant
implications of aberrant DNA methylation come from its tight connection with the immune cells system.
To date, immune-checkpoint inhibitors (ICI) have shown striking results in selected cancer types, although
only a minority of patients are sensitive to these drugs. De novo DNA methylation has been recently
reported to have a central role in maintaining a T cell exhaustion status that contributes to resistance to
ICI treatment . On the other hand, previous studies demonstrated that DNA demethylating drugs can
[173]
enhance CTLA-4 blockade-mediated T cell responses . Moreover, treatment of epithelial cancer cell lines
[174]
(including CRC cell lines) with demethylating agents, i.e. 5-azacitidine, has been reported to promote a
significant enrichment of immunomodulatory pathways . As a possible explanation, cryptic transcription
[175]
of thousands of treatment-induced non-annotated transcriptional start sites (TINATs) may contribute to
cancer immunogenicity through the translation of novel potential antigenic proteins, as recently shown
by Brocks and colleagues in their work exploring DNA methyltransferases inhibitors (DNMTi) treatment
[176]
consequences on epigenetic and genome-wide transcription . Overall, this growing evidence supports a
strong immunomodulatory effect of DNA demethylating agents in cancer cells, and the rationale to combine
these drugs with immunotherapy in cancer patients. Based on these premises, a deeper understanding of
the interplay between epigenetic modifications, cancer cells and immune cells could reveal novel potential
strategies to enhance ICI treatment efficacy and overcome primary and acquired resistance mechanisms to
immunotherapy.

Finally, aberrant DNA methylation may exert a direct effect modulating well-established molecular pathways
in CRC. Notably, EGFR promoter DNA methylation has been reported to occur in 58% of primary colon