We conducted this study to examine the relations of the class III histone deacetylase (HDAC) SIRT1 with the CpG island methylator phenotype (CIMP), related molecular events and patient outcome in colorectal cancer. Molecular correlates with SIRT1 activation may be important for better understanding of epigenetic and epigenomic aberrations during the carcinogenic process. We have found that SIRT1 expression is significantly associated with CIMP-high and microsatellite instability (MSI). Moreover, SIRT1 expression is significantly associated with the CIMP-high MSI-high phenotype, independent of other clinical and molecular variables. In contrast, SIRT1 expression is not related with global DNA methylation level as measured in LINE-1 repetitive sequence. Our data support the hypothesis that SIRT1 is related with methylation at individual CpG islands, but not with global DNA methylation, in colorectal cancer.
Studying molecular changes is important in cancer research.47–53
To measure DNA methylation, we utilized real-time PCR (MethyLight technology) for DNA methylation at the 8 CIMP-specific loci 30
and 8 other CpG islands. We also utilized Pyrosequencing to measure LINE-1 methylation that has been correlated with cellular 5-methylcytosine level (i.e., genome-wide DNA methylation level).41
Our resource of a large number of colorectal cancers derived from the two prospective cohort studies has enabled us to precisely estimate the frequency of colorectal cancers with a specific molecular feature (such as SIRT1 overexpression, CIMP-high, MSI-high, etc.). The large number of cases has also provided a sufficient power in our multivariate logistic regression analysis and survival analysis.
Recent studies have reported that up-regulation of SIRT1 may prolong cell survival through multiple mechanisms, and play an important role in the regulation of epigenetic alterations.1, 2, 16, 17
In addition, SIRT1 silences genes through deacetylation of the histone residue, H4K16.8, 54, 55
Our data are likely important, because no study has demonstrated the relationship between SIRT1 and CIMP in human colorectal cancer. On the other hand, our data do not support a direct link between SIRT1 and genome-wide DNA methylation level. SIRT1 has been reported to localize to the promoters of several aberrantly silenced tumor suppressor genes in colon cancer cells, in which CpG islands are hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed.16
These experimental data are consistent with our data of the positive association between SIRT1 and CIMP-high, but no significant relation between SIRT1 and genome-wide DNA methylation level.
With regard to the relationship between MSI and HDACs, a recent study has reported the presence of a truncating mutation in HDAC2 (class I) in MSI-high colorectal cancers.56
However, no study has reported the relation between SIRT1 and MSI. It is important to analyze both CIMP and MSI to decipher the interrelationship between SIRT1, CIMP and MSI. In the current study, we have shown the significant association between SIRT1 and the CIMP-high MSI-high subtype, and it is particularly strong among BRAF
-mutated cancers. Further studies are necessary to elucidate the relation between SIRT1 activity, BRAF
, MSI and CIMP.
Recent studies have reported that epigenetic inactivation of HIC1
results in up-regulation of SIRT1, which deacetylates p53, and that SIRT1 down-regulates p53 through histone deacetylation.15, 16
In addition, SIRT1 has been reported to down-regulate β-catenin through deacetylation and suppresses its ability to facilitate transcription and cell proliferation.17
However, we failed to show associations of SIRT1 with HIC1
methylation, p53 expression and β-catenin activation. Possible explanations include a difference in patient cohorts, and false positive/negative results in immunohistochemistry. In particular, the presence of poorly preserved tissue specimens might show false negative results on either SIRT1 or β-catenin, which might obscure the inverse relation between nuclear β-catenin and SIRT1 expression. Nonetheless, our classification of SIRT1 status appeared to be valid, since we were able to show the strong association between SIRT1 and the CIMP-high MSI-high subtype.
SIRT1 has been reported to be induced by calorie restriction in multiple tissues of mammals.3–5
Moreover, at the cellular level, SIRT1 may facilitate this process by regulating energy metabolism.8
Although we have shown no significant relation between patient body mass index and SIRT1 expression, we have shown the relation between SIRT1 and FASN. These results suggest that SIRT1 may cooperate with FASN in regulating energy metabolism in cancer cells.
Many studies have reported anti-tumor effects of HDAC inhibitors, DNA methyltransferase inhibitors and histone lysine demethylases.1, 2, 57, 58
Interestingly, a recent study has reported that blocking SIRT1 function synergizes with both promoter demethylation and inhibition of class I and II HDACs for gene reactivation.16
Moreover, this inhibition of SIRT1 leads to gene reactivation even with retention of DNA methylation.16
These results suggest new directions for targeting reversal of abnormal gene silencing and demonstrate the importance of ongoing and future studies, which may lead to the eventual translation into clinical practice. In the current study, we have demonstrated a significant association between SIRT1 and CIMP-high MSI-high colorectal cancer. These findings may indicate that therapies targeting SIRT1 may be particularly useful for this CIMP-high MSI-high subtype of cancer.
In conclusion, SIRT1 expression is significantly associated with CIMP-high MSI-high status, particularly in the presence of BRAF mutation. Our data also indicate that SIRT1 is related with DNA methylation in gene-specific CpG islands, rather than global DNA methylation level. Considering that SIRT1 is a promising target of chemotherapy and chemoprevention, our findings may have considerable clinical implications.