To our knowledge, this is the first study to comprehensively examine the association between dietary intake and promoter methylation of genes related to head and neck cancer. These novel findings suggest diet is significantly associated with epigenetic events that occur in head and neck cancer. While previous studies have indicated deficiencies of folate, vitamin B12 and vitamin A are associated with increased risk of developing head and neck cancer5,19-21
as well as with poorer prognosis in patients diagnosed with the disease,22
our study has shown a decrease in tumor suppressor methylation in the highest quartile of vitamin B12 and vitamin A intake. Epigenetic dysregulation, such as the silencing of tumor suppressor genes via promoter hypermethylation, is a major event in malignant disease.
Though the underlying biological mechanism related to how folate and vitamin B12 deficiencies may be associated with DNA hypermethylation has not been elucidated, we have two suggested hypotheses. First, folate and vitamin B12 play important roles in the synthesis of DNA and DNA damage repair.23-25
Increases in DNA methylation have been observed in an experimental model inducing in vitro DNA damage, in the form of DNA strand breaks, and homology directed DNA repair.26
An increased rate of DNA strand breaks was also observed in both animal models and cell culture models of folate deficiency,27,28
potentially providing a mechanism that links folate deficiency, DNA strand breaks, and DNA methylation. Second, while the DNA methylation changes that occur in cancer have been well characterized, there is also interest in describing the DNA methylation changes that occur with aging, as these changes could reflect what occurs in the early stages of carcinogenesis. A recent study has described that DNA methylation of two genes, ERα
, in colon tissue increases with age and also with folic acid supplementation status.29
In our study, we not only observed a decrease in tumor suppressor methylation in the highest quartile of vitamin A, vitamin B12 and food folate intake, but also a trend toward increased tumor suppressor methylation in the individuals who consumed the highest levels of refined grains. During the refinement process these grains are fortified with folic acid, niacin, riboflavin, thiamin and iron. It is unclear how these nutrients may be associated with increased DNA methylation and more research is necessary.. The relative bioavailability of these nutrients could modulate the normal stochastic changes in methylation that occur due to aging and may be reflected in tumor DNA methylation.
Other studies support our finding that increased consumption of folate and folate-rich foods are associated with decreased methylation of genes associated with tumor suppression. Another study conducted in head and neck cancer patients reported increased methylation of p16INK4a
, a gene involved in cell cycle regulation that is commonly lost or silenced in head and neck cancer via epigenetic mechanisms, in those consuming the lowest levels of folate compared with the highest levels.30
Based on our findings that folate intake was inversely associated with tumor suppressor DNA methylation, it is not surprising that high cruciferous vegetable consumption was associated with lower methylation levels, given this food group is a rich source of folate. However, it is interesting to note that cruciferous vegetables are also rich sources of glucosinolates such as sulforphane and indole-3-carbinol, both of which are believed to exhibit anti-cancer activities. Though there is little evidence suggesting involvement of sulforaphane in DNA methylation, this compound has been shown to act as a histone deacetylase (HDAC) inhibitor. HDACs have been shown to be widely overexpressed in a number of cancers and are involved in gene silencing by inducing a more condensed chromatin state, leaving DNA less accessible for transcription.31
HDAC inhibitors, such as sulforaphane, have been shown to be promising cancer therapies because of their ability to induce expression of epigenetically silenced genes.32
The finding that vitamin A intake is significantly associated with decreased DNA methylation in HPV(-) tumors is unexpected, considering vitamin A does not play a direct role in the one-carbon metabolism pathway. However, it is possible vitamin A can alter DNA methylation by influencing the availability and activity of methyltransferases. Two studies have reported rats treated with all-trans
-retinoic acid (ATRA), a vitamin A metabolite, displayed increased levels and activity of glycine N
-methyltransferase (GNMT), the enzyme involved in the conversion of S-adenosyl methionine (SAM) to S-adenosylhomocysteine (SAH).33,34
Increased GNMT activity led to a reduction of SAM, thus potentially resulting in a reduction of methylation. Similarly, Das et al. reported demethylation of 402 gene promoters in neuroblastoma cells treated with ATRA. These cells also exhibited downregulation of the methyltransferases, DNMT1
, which the authors proposed as the potential mechanism by which ATRA exerts its’ effect on DNA methylation.35
The GSEA findings of this study confirm the previous work of our group and others describing the differences in tumor biology between HPV(+) and HPV(-) HNCs.36,37
In this study, we identified that gene sets involved in immune function, including cytokine production and antigen processing and presentation, were significantly enriched based on DNA methylation differences in HPV(+) tumors from individuals with high and low levels of vitamin B12 intake. There was no consistent KEGG or Gene Ontology Biological Processes enriched in HPV(-) tumors, based on either vitamin A or vitamin B12 intake. A positional enrichment analysis identified the same chromosomal region, chromosome 12p12, as differentially methylated in HPV(+) tumors based on vitamin B12 intake as HPV(-) tumors based on vitamin A intake. This region has previously been identified as a candidate tumor suppressor region in head and neck and other cancers,38
and may represent a region of the genome that is more labile to the epigenetic influences of dietary intake.
Results of this preliminary study should be interpreted in light of several limitations. Our small sample size poses analytic limitations in detecting associations of interest and the complexity of statistical tests used. Although we have adjusted for several covariates, there are no doubt other unmeasured sources of confounding we were not able to consider. The FFQ is susceptible to measurement error and potential unquantified systematic biases that may have led to misclassification of dietary intake. The FFQ was designed to assess usual dietary intake over the course of the year prior to diagnosis with HNC. Thus we cannot make any conclusions about the association between lifetime dietary exposures and tumor DNA methylation. While the Goldengate Cancer Panel provides a comprehensive measurement of methylation across 1505 CpG sites in promoter regions of genes with known roles in cancer, this technology does not provide an unbiased assessment of the epigenome. Finally, due to these limitations, the findings of this study should be validated in another set of tumors from similar individuals.
In conclusion, dietary intake of micronutrients involved in one-carbon metabolism such as folate and vitamin B12, as well as other micronutrients such as vitamin A, may be significant regulators of epigenetic events occurring in head and neck cancer. These findings have potential clinical implications for the treatment of head and neck cancer. The idea that dietary interventions could potentially reprogram the epigenome in such a way as to optimize the likelihood of positive disease outcomes is appealing. Further research is necessary to better understand the complex role these micronutrients play in regulating the epigenetic process, as well as how nutrients may differentially regulate this process according to HPV status. Such studies could give rise to highly specific randomized controlled trials and ultimately to the development of individualized medical nutrition therapy regimens that may improve prognosis in the head and neck cancer population.