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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Int J Cancer. Author manuscript; available in PMC 2010 May 1.
Published in final edited form as:
PMCID: PMC2692263
NIHMSID: NIHMS99332

The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: A possible molecular basis for the site-specific cancer risk modification

Abstract

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer while it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one-carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one-carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation.

Keywords: MTHFR C677T polymorphism, folate, colon cancer, breast cancer, DNA methylation, uracil misincorporation

Introduction

Intracellular folate homeostasis depends on 5,10-methylenetetrahydrofolate reductase (MTHFR) that catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate (5,10-methyleneTHF) to 5-methyltetrahydrofolate (5-methylTHF) (Figure 1) 1, 2. The substrate 5,10-methyleneTHF is the methyl donor for the nonreversible methylation of deoxyuridine-5-monophosphate to deoxythymidine-5-monophosphate (thymidylate) 1. 5,10-MethyleneTHF can also be oxidized to 10-formylTHF for de novo purine synthesis 1, 2. Therefore, 5,10-methyleneTHF is critical in maintaining the balance of the nucleotide pool for DNA synthesis 1, 2. 5-MethylTHF, the product of MTHFR, provides the methyl group for remethylation of homocysteine to methionine, thereby ensuring the provision of S-adenosylmethionine (SAM) necessary for most biological methylation reactions including that of cytosine located within the cytosine-guanine (CpG) dinucleotide sequences of DNA (Figure 1) 1, 2.

Figure 1
Simplified scheme of the role of 5,10-methylenetetrahydrofolate reductase (MTHFR) in folate metabolism and one-carbon transfer reactions involved in DNA synthesis and biological methylation reactions, including that of DNA. MTHFR catalyzes the irreversible ...

The MTHFR C677T polymorphism, which results in an alanine to valine substitution 3, 4, is a common variant with an allele frequency of about 35% in the general North American population 3, 5, and occurs frequently among Caucasian and Asian populations, with rates of ~12%-15% for individuals who are homozygous for the variant and up to 50% for individuals who are heterozygous 2. Although not uniformly consistent, this polymorphism has been shown to modify risk for several cancers in a site-specific manner 1, 2. It appears to decrease the risk of colorectal cancer, hepatocellular carcinoma, cervical cancer, and certain leukemias and lymphomas 1, 2. In contrast, this polymorphism seems to increase the risk of cancer of the breast, endometrium, esophagus, stomach, pancreas, and bladder 1, 2. For some cancers, the cancer risk modification conferred by this polymorphism is further modified by the status of folate and nutrients involved in one-carbon and folate metabolism 2, 6, 7. For colorectal cancer, epidemiologic evidence indicates a protective effect of this polymorphism in individuals with adequate or high status of folate and nutrients involved in one-carbon metabolism 7-9. In those with an inadequate status of folate and related nutrients, the protective effect conferred by this polymorphism was diminished and in some cases, an increased risk of colorectal cancer was observed 7. However, this perceived diminished protection conferred by this polymorphism with increasingly low folate status has not been unequivocally confirmed and it has been suggested that this may be an analytical artefact 10. For breast cancer, this polymorphism seems to either increase the risk 11, 12 or have no effect 13.

The mechanisms by which the MTHFR C677T polymorphism could modulate the risk of colorectal and breast cancers have not yet been elucidated. This polymorphism is associated with reduced MTHFR activity 3 and increased thermolability of MTHFR 3, 4 in vitro, which lead to decreased 5-methylTHF and an accumulation of 5,10-methyleneTHF in red blood cells 14. Recent biochemical and structural studies of Escherichia coli and human MTHFR have revealed that the MTHFR C677T polymorphism allows a faster dissociation of a critical stabilizing cofactor, FAD (flavin adenine dinucleotide or a coenzyme form of riboflavin), from the variant MTHFR compared with the wild-type MTHFR, resulting in thermolability and significantly reduced MTHFR activity 15, 16. Adequate folate or riboflavin protected MTHFR from the loss of FAD cofactor, thereby ensuring functional MTHFR activity 15, 16. Under the conditions of high folate or riboflavin, the enzyme kinetics of the variant MTHFR were similar to those of the wild-type enzyme 15, 16. This infers that only at low folate or riboflavin concentrations will the functional impact of the MTHFR C677T variant become significant. Indeed, the MTHFR C677T polymorphism was associated with increased plasma homocysteine (a sensitive inverse indicator of folate status) concentrations 5, 17-19 and genomic DNA hypomethylation 20-24 in lymphocytes only in individuals with low folate or riboflavin status.

Based on these biochemical consequences, one proposed mechanism suggests that when the dietary supply of folate and related nutrients is high, individuals with this polymorphism might be at reduced risk of cancer because higher intracellular levels of 5,10-methyleneTHF might prevent imbalances of the nucleotide pool during DNA synthesis, thereby ensuring DNA replication with a high fidelity (Figure 1) 25. Furthermore, with high intakes of folate and related cofactors, the flux of 5,10-methyleneTHF to 5-methylTHF would function at its full capacity and hence, individuals with this polymorphism would have adequate levels of SAM for optimal DNA methylation (Figure 1) 25. When intakes of folate and related nutrients are low, the reduced stability of the variant MTHFR results in deactivation of the MTFHR enzyme and therefore decreased flux of 5,10-methyleneTHF towards the methionine cycle pathway at a higher threshold of folate availability. This would maintain the availability of 5,10-methyleneTHF and reduced the likelihood of compromised DNA synthesis and consequent nucleotide pool imbalance (Figure 1) 25. In this instance, however, DNA methylation might be affected because of reduced levels of 5-methylTHF resulting from an insufficient supply from the diet and reduced flux of 5,10-methyleneTHF to the methionine cycle due to the decreased stability of the variant MTHFR enzyme (Figure 1) 25. DNA damage, genomic instability, and impaired DNA repair resulting from a nucleotide pool imbalance are important mechanisms of carcinogenesis 26, 27. Both genomic DNA hypomethylation and gene-specific promoter CpG island hypermethylation are also important epigenetic mechanisms of carcinogenesis 28. However, these purported functional effects of the MTHFR C677T polymorphism have not yet been clearly demonstrated in target organs.

We have previously reported the generation of an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast cancer cells with predictable functional consequences 29. Using this in vitro model, we determined the effect of the MTHFR C677T polymorphism on intracellular methionine cycle intermediates, homocysteine, DNA methylation and uracil misincoporation to elucidate mechanisms by which this polymorphism might modulate colon and breast cancers in cells derived from the target organs.

Materials and Methods

The in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast cancer cells

We have previously generated and functionally characterized an in vitro model of the MTHFR 677T mutation in human HCT116 colon and MDA-MB-435 breast cancer cells stably transfected with the wild-type and mutant 677T MTHFR cDNAs 29. In this model, the MTHFR 677T mutation was associated with decreased MTHFR activity, increased MTHFR thermolability, decreased intracellular 5-methylTHF and increased intracellular 5,10-methyleneTHF, accelerated cellular growth rate, increased thymidylate synthase activity, and significant changes in chemosensitivity to 5-fluorouracil and methotrexate in a predictable manner compared with controls 29. Parental HCT116 and MDA-MB-435 cell lines are both heterozygous for the MTHFR C677T polymorphism 29.

Several clones expressing the wild-type and 677T mutant from each cell line were selected at random and two independent clones of each construct were selected for analyses. For all analyses except for genomic DNA methylation, cells expressing the wild-type and mutant 677T MTHFR were grown at 37 °C in 5% CO2 in standard RPMI 1640 medium (Invitrogen, Gaithersburg, MD) containing 2.3 μmol/L folic acid and 100 μmol/L L-methionine supplemented with 10% fetal bovine serum and neomycin at 500 μg/mL. Cells were harvested at 80% confluence and were processed for subsequent analyses. For genomic DNA methylation, cells expressing the wild-type and mutant 677T MTHFR were grown for 9 d in standard RPMI 1640 medium containing 2.3 μmol/L folic acid or in RPMI 1640 medium free of folic acid supplemented with 5 nmol/L or 20 nmol/L folic acid (Sigma, Oakville, ON, Canada) to determine MTHFR-folate interactions in modifying genomic DNA methylation. Data from several replicate experiments using two independent clones of each construct were similar and thus the data from one experiment are presented.

Determination of intracellular methionine cycle intermediates

SAM and S-adenosylhomocysteine (SAH) were determined using reversed-phase HPLC as described 30. All analyses were performed in triplicate and repeated using three independent cell lysates.

Determination of intracellular homocysteine concentrations

Intracellular homocysteine concentrations were measured in three different states: the native and post-L-methionine (1 mmol/L) and post-D, L-homocystiene loading (500 μmol/L) states. Cells were grown to confluence in RPMI 1640 medium and were exposed to 1 mmol/L L-methionine or 500 μmol/L D, L-homocysteine for 4 h. Cells were washed three times in RPMI 1640 medium and three times in PBS. Cells were lysed in water by three freeze/thaw cycles and cellular debris removed by centrifugation. Total homocysteine was determined in cellular lysates using the IMx System (Abbott Laboratories, Mississauga, Ontario, Canada) as described 31 and normalized to total sample soluble protein concentration. We initially used 5 mmol/L D, L-homocysteine loading in HCT116 cells expressing endogenous MTHFR, which produced a 20-30 fold increase in intracellular homocysteine concentrations over the baseline value, to establish reliable assay conditions and sensitivity of the assay. The IMx system has been successfully utilized to measure intracellular homocysteine concentrations in human T24/83 transitional bladder carcinoma cells, human aortic smooth muscle cells, primary human umbilical vein endothelial cells, and human HepG2 hepatocarcinoma cells 31, 32. To increase intracellular homocysteine levels in these culture cells, we and others have applied exogenous D, L- or L-homocysteine in concentrations ranging from 50 μmol/L to 5 mmol/L without impairing cell viability 31-34. All analyses were performed in quadruplicate and repeated using two independent cell lysates.

DNA methyltransferase (DNMT) activity assay

Total cellular CpG DNMT activity was measured by incubating cell lysates containing10 μg of protein with 0.5 μg of poly[d(I-C).d(I-C)] template (Amersham Pharmacia Biotech, Piscataway, NJ) and 111 kBq [3H]-SAM (NEN Life Sciences, Boston, MA) for 2 h at 37 °C as described 35. Each reaction was performed in triplicate and the assay was repeated three times.

Genomic DNA Isolation

Total genomic DNA was extracted by a standard technique using proteinase K followed by organic extraction 36. The size of DNA estimated by agarose gel electrophoresis was >20 kb in all instances. No RNA contamination was detected on agarose gel electrophoresis. The final preparations had a ratio of A260:A280 between 1.8 and 2.0. The concentration of each DNA sample was determined as the mean of three independent spectrophotometric readings.

Genomic DNA Methylation

The methylation status of CpG sites in genomic DNA was determined by the in vitro methyl acceptance assay using [3H-methyl]SAM (NEN Life Sciences) as a methyl donor and a prokaryotic CpG DNMT, Sss1 (New England Biolabs, Beverly, MA), as described 35. The manner in which this assay is performed produces a reciprocal relationship between the endogenous DNA methylation status and exogenous [3H-methyl] incorporation. The extent of genomic DNA methylation was determined in cells with varying constructs grown for 9 d in standard RPMI 1640 medium containing 2.3 μmol/L folic acid or in RPMI 1640 medium free of folic acid supplemented with 5 nmol/L or 20 nmol/L folic acid (Sigma, Oakville, ON, Canada). All analyses were performed in quadruplicate and repeated using two independent cell lysates.

Quantitative analysis of methylation levels in CpG-rich regions of the genome

The bisulfite modification-based MethyLight assay was used to quantitate methylation at 70 CpG-rich regions, mostly CpG islands overlapping the 5' or promoter regions, of 67 genes including those that are epigenetically regulated in colorectal carcinogenesis as described 37, 38. The complete list of these genes with the primers and probes has been published 37, 38. The percentage of fully methylated molecules at a specific CpG-rich gene region was calculated by dividing the MethyLight signal for the given gene by that for βactin in each sample DNA and then diving that ratio by the analogous GENE: βactin for in vitro-methylated sperm DNA and multiplying by 100.

Western blot analysis

STK11 (serine/threonine kinase 11) protein expression was determined by standard Western blot analysis as described 29, using a rabbit polyclonal antibody raised against a peptide sequence spanning amino acids 1 - 100 of human STK11 (Abgent, San Diego, California) at a dilution of 1:300.

Uracil misincorporation into DNA

Increased uracil content in DNA reflects imbalance of the deoxyribonucleotide pool, especially reduced thymidylate 39. Uracil in DNA was measured by a GC/MS method as described 40. This assay was performed in three independent cell lysates.

Statistics

Comparisons between cells expressing mutant and wild-type MTHFR were determined using Student's t-test. All significance tests were 2-sided and were considered significant at P < 0.05. Results are expressed as mean ± SD. Statistical analyses were performed using SAS, version 8 (SAS Institute, Cary, NC).

Results

Effect on intracellular methionine cycle intermediates

Intracellular SAM and SAH concentrations were 27% and 11% lower, respectively, in HCT116 cells expressing the mutant 677T MTHFR than in cells expressing the wild-type MTHFR (P < 0.0001 and P=0.0059 respectively) (Table 1). The SAM to SAH ratios were 29% lower in HCT116 cells expressing the mutant 677T MTHFR than in cells expressing the wild-type MTHFR (P < 0.0001) (Table 1).

Table 1
Intracellular SAM and SAH concentrations and SAM:SAH ratios in HCT116 colon and MDA-MB-435 breast cancer cells expressing the mutant 677T or wild-type (WT) MTHFR1

In contrast, intracellular SAM and SAH concentrations were 74% and 16% higher, respectively, in MDA-MB-435 cells expressing the mutant 677T MTHFR than in cells expressing the wild-type MTHFR (P < 0.0001 and P=0.023, respectively) (Table 1). The SAM to SAH ratios were 50% higher in MDA-MB-435 cells expressing the mutant 677T MTHFR than in cells expressing the wild-type MTHFR (P=0.0006) (Table 1).

Effect on intracellular homocysteine

Intracellular homocysteine concentrations were significantly higher in HCT116 cells expressing the mutant 677T MTHFR than in cells expressing the wild-type MTHFR in the native state and after L-methonine or D, L-homocysteine loading (P < 0.02) (Table 2).

Table 2
Intracellular homocysteine concentrations in HCT116 colon and MDA-MB-435 breast cancer cells expressing the mutant 677T or wild-type (WT) MTHFR1

In contrast, intracellular homocysteine concentrations tended to be lower in the native state (P=0.062) and after L-methionine loading (P=0.095) but higher after D, L-homocysteine loading (P=0.07) in MDA-MB-435 cells expressing the mutant 677T MTHFR than in cells expressing the wild-type MTHFR (Table 2).

Effect on DNMT activity

Total cellular CpG DNMT activity was significantly higher (by 1.4 - 2.0 fold) in both HCT116 and MDA-MB-435 cells expressing the mutant 677T MTHFR than in corresponding control cells expressing the wild-type MTHFR (P < 0.0001 and P=0.014, respectively) (Table 3).

Table 3
The effects of the MTHFR 677T mutation on DNMT activity, genomic DNA methylation and uracil misincorporation in HCT116 colon and MDA-MB-435 breast cancer cells1

Effect on genomic DNA methylation

The extent of genomic DNA methylation was determined in medium containing three different folic acid concentrations, 2.3 μmol/L (normal medium concentration representing an excessively high level), 20 nmmol/L (representing a sufficient level) and 5 nmol/L (representing a low level). Although the extent of genomic DNA methylation in HCT116 cells expressing the mutant 677T MTHFR was 6.4% and 4.8% higher than in cells expressing the wild-type MTHFR at 2.3 μmol/L and 20 nmol/L folic acid, respectively (P=0.0018 and P=0.038, respectively), at 5 nmol/L folic acid, the extent of genomic DNA methylation in HCT116 cells expressing the mutant 677T MTHFR was 8.2% lower than in cells expressing the wild-type MTHFR (P=0.001) (Table 3).

In contrast, the degree of genomic DNA methylation was 16.4% and 11.0% lower in MDA-MB-435 cells expressing the mutant 677T MTHFR than in cells expressing the wild-type MTHFR at 2.3 μmol/L and 20 nmol/L folic acid, respectively (P=0.0002 and P < 0.0001, respectively). However, at 5 nmol/L folic acid, no significant difference in the extent of genomic DNA methylation between the two constructs was observed (p=0.24) (Table 3).

Effect on promoter CpG island methylation

In HCT116 cells, no significant difference in the degree of promoter CpG island methylation was observed between HCT116 cells expressing the mutant MTHFR 677T and those expressing the wild-type MTHFR in most of the CpG islands loci overlapping the 5' or promoter regions of the 67 genes screened. Furthermore, the degree of promoter CpG islands of the selected genes in untransfected HCT116 cells was similar to that of HCT116 cells transfected with the wild-type and mutant 677T MTHFR cDNAs.

However, the promoter CpG island of the STK11 was 54-57% methylated in HCT116 cells expressing the wild-type MTHFR compared with 4-6% in those expressing the mutant MTHFR 677T. In untransfected HCT116 cells, this CpG locus was 30% methylated. However, STK11 protein expression was not significantly different between cells expressing the wild-type MTHFR and cells expressing the mutant MTFHR 677T (data not shown), suggesting no significant functional effect of the observed hypomethylation of the promoter CpG island of the STK11 gene associated with the MTHFR 677T mutation.

Effect on uracil misincorporation

HCT116 cells expressing the mutant MTHFR 677T had 31% lower, albeit not statistically significant, uracil misincorporation compared with cells expressing the wild-type MTHFR (P=0.07) (Table 3). In contrast, MDA-MB-435 cells expressing the mutant MTHFR 677T had 35% higher, albeit not statistically significant, uracil misincorporation compared with cells expressing the wild-type MTHFR (P=0.08) (Table 3).

Discussion

In the present study, we took an epidemiologic observation to a molecular level in an attempt to elucidate biologically plausible mechanisms by which the MTHFR C677T polymorphism could influence the risk of colorectal and breast cancers in a site-specific manner using a well-established in vitro model of the MTHFR C677T mutation 29. This is the first study to show the functional effects of the MTHFR 677T variant in cells derived from the target organs of interest in an in vitro model of the MTHFR C677T polymorphism that circumvents the limitations associated with animal models and clinical studies. We demonstrated that the effects of the MTHFR 677T mutation on intracellular methionine cycle intermediates, homocysteine, DNA methylation, and uracil misincorporation are cell-specific, thereby providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation.

The observed significant reduction in intracellular SAM concentrations and SAM to SAH ratios and the significant increase in intracellular homocysteine concentrations associated with the MTHFR 677T mutation in HCT116 cells suggest that the culture conditions induced a sufficient degree of intracellular folate depletion, although the culture medium contained an abundant amount of folic acid (2.3 μmol/L). Previous in vitro 15, 16, 41 and in vivo 5, 17-23 studies have suggested that the functional impact of the MTHFR C677T variant becomes apparent only in conjunction with low folate status. Indeed, intracellular folate concentrations of HCT116 cells transfected with the mutant and wild-type MTHFR are 24-72% lower than those of untransfected HCT116 cells grown in the same culture medium 29, 35, 42. This may be related to the observation that HCT116 cells transfected with the wild-type or mutant MTHFR demonstrated accelerated cellular growth rate compared with untransfected cells in the same culture medium 29. The rapid proliferation might have reduced intracellular folate availability even in a medium containing abundant folate.

The observed significant reduction in intracellular SAM concentrations and SAM to SAH ratios and the significant increase in intracellular homocysteine concentrations associated with the MTHFR 677T mutation in HCT116 cells are consistent with the biochemical consequence of decreased 5-methylTHF and hence, compromised remethylation of homocysteine to methionine (Figure 1). Furthermore, decreased intracellular SAM concentrations result in relatively unstimulated cystathionine β-synthase in the transsulfuration pathway and consequent decreased homocysteine catabolism, resulting in homocysteine accumulation (Figure 1) 43. However, the observed significant reduction in intracellular SAH concentrations is unexpected and cannot be readily explained. The conversion of SAH to homocysteine is catalyzed by SAH hydrolase and the equilibrium of this reaction actually favors the formation of SAH and is driven forward by the removal of the products 43. Therefore, a significant intracellular homocysteine accumulation resulting from the MTHFR 677T mutation should have resulted in increased intracellular SAH concentrations.

In HCT116 cells, the MTHFR 677T mutation was associated with a significant increase in DNMT activity. This may be a compensatory response to the observed decreased intracellular SAM concentrations to maintain a critical level of DNA methylation. Compensatory upregulation of DNMT in response to decreased SAM resulting from folate or methyl group donor deficiency has been previously reported 28, 42, 44, although some studies showed downreguation of DNMT at the translational or posttranslational level 35, 45. However, an alternative explanation for the increased DNMT activity is related to the observation that HCT116 cells expressing the mutant 677T MTHFR demonstrate accelerated cellular growth rate and increased thymidylate synthase activity compared with those expressing the wild-type MTHFR 29. Since DNMT expression peaks during S-phase 46, 47, it would be expected that DNMT expression would be higher in HCT116 cells expressing the mutant 677T MTHFR due to their more proliferative phenotype. Also, DNMT activity is expected to be increased because of the unexpected decrease in intracellular SAH concentrations in HCT116 cells expressing the mutant 677T MTHFR. SAH is a potent inhibitor of most SAM-dependent methyltransferases 43.

Parallel to the observed increased DNMT activity, the MTHFR 677T mutation in HCT116 cells was associated with a significant increase in DNA methylation at sufficient and very high folate concentrations. However, this observation has not been demonstrated in previous in vitro and in vivo studies and thus warrants confirmation to establish that the prevention of genomic DNA hypomethylation might be a mechanism by which the MTHFR 677T mutation may protect against colorectal cancer in the setting of adequate or high folate status. At a low folate concentration, the MTHFR 677T mutation was associated with a significant reduction in DNA methylation, thereby corroborating the prior observation that the MTHFR C677T polymorphism is associated with genomic DNA hypomethylation in human lymphocytes only in conjunction with low folate status 20-23. However, given the observation that even under conditions of low folate status, the TT genotype is associated with a slightly lower risk of colorectal cancer than the wild-type genotype 10, genomic DNA hypomethylation is not a likely mechanism by which the MTHFR 677T mutation increases the risk of colorectal cancer in the setting of folate insufficiency. The observed changes in DNA methylation in HCT116 cells were very modest, albeit significant, and their functional ramifications need to be characterized. Given the fact that it is extremely difficult to alter genomic and site-specific DNA methylation in HCT116 cells 35, 48, however, the observed changes in DNA methylation in HCT116 cells may be an underestimation of that exiting in normal colonic epithelial cells.

The MTHFR 677T mutation generally had no effect on methylation at CpG islands loci of the 67 genes screened, including critical tumor suppressor and cancer-related genes that are epigenetically regulated in colorectal carcinogenesis, except for hypomethylation of the promoter CpG island of the STK11 gene, which encodes a serine-threonine kinase that modulates cellular proliferation, controls cell polarity, and plays an important role in responding to lower cellular energy levels 49. Germline mutations of STK11 are reported in up to 70-80% of patients with Peutz-Jeghers syndrome, which is associated with a markedly increased risk of malignancy including colorectal cancer 49. However, the observed hypomethylation of the promoter CpG island of the STK11 gene was not associated with a significant increase in STK11 protein expression.

Unexpectedly, the MTHFR 677T mutation was associated with a significant increase in intracellular SAM and SAH concentrations and the SAM to SAH ratios and a significant decrease in intracellular homocysteine concentration in MDA-MB-435 cells. These paradoxical observations cannot be readily explained but may be in part related to the presence and relative activity of several enzymes involved in methionine cycle that are inherently different between HCT116 and MDA-MB-435 cells and that may respond differently to the MTHFR 677T mutation. In this regard, some studies have reported cell-specific, and in some cases paradoxical, effects of folate on methionine cycle intermediates and enzymes regulating this process 35, 42.

Similar to HCT116 cells, the MTHFR 677T mutation was associated with a significant increase in DNMT activity in MDA-MB-435 cells. Despite increased DNMT activity, however, the MTHFR 677T mutation was associated with a significant decrease in DNA methylation at sufficient and very high folate concentrations. At a low folate concentration, the MTHFR 677T mutation had no effect on DNA methylation. Our data suggest a MTHFR 677T mutation-folate interaction in modifying genomic DNA methylation similar to that observed in HCT116 cells. Our data also suggest a biologically plausible mechanism by which the MTHFR 677T mutation may increase the risk of breast cancer: the induction of genomic DNA hypomethylation.

Although not uniformly consistent 50, evidence suggests that the MTHFR C677T polymorphism induces genomic DNA hypomethylation in human lymphocytes in conjunction with low folate status 20-23. However, these observations made in lymphocytes cannot be extrapolated to target organs of interest. Published studies in cancer cell lines and human colorectal biopsies have reported no effect of the MTHFR C677T polymorphism on CpG islands methylation of a small number (9-15) of tumor suppressor genes 51-53. Mthfr-/- and Mthfr+/-mice had significantly decreased SAM and increased SAH concentrations and decreased genomic DNA methylation compared with Mthfr+/+ mice in a organ-specific manner 54, 55. However, these functional effects of MTHFR deficiency were not determined in the colorectum or mammary glands.

Our data concerning the effect of the MTHFR 677T mutation on uracil misincorporation did not reach statistical significance. This might reflect the ongoing base excision repair of uracil misincorporated into genomic DNA, thereby potentially masking the full impact of nucleotide imbalances. Nevertheless, the trend suggests that this might be also cell-specific. Consistent with the expected biochemical ramification, the MTHFR 677T mutation was associated with a 31% decrease in uracil misincorporation in HCT116 cells. Decreased MTHFR activity associated with this mutation results in an accumulation of intracellular 5,10-methyleneTHF, thereby providing a greater supply of this substrate for enhanced thymidylate synthesis and hence, decreased uracil misincorporation (Figure 1). Furthermore, increased 5,10-methyleneTHF would result in enhanced purine synthesis (Figure 1). The net effect of an accumulation of 5,10-methyleneTHF would be DNA replication with a high fidelity, stable DNA integrity, and decreased mutagenesis. Unexpectedly, the MTHFR 677 mutation was associated with a 36% increase in uracil misincorporation in MDA-MB-435 cells. This is not consistent with the known biochemical consequence of the MTHFR 677T mutation and cannot be readily explained. Again, this may be related to as yet undetermined responses of methonine cycle enzymes to MTHFR inhibition unique to MDA-MB-435 cells. Increased uracil misincorporation results in double strand DNA breaks leading to genomic instability and enhanced neoplastic transformation 26.

In vitro studies using immortalized human lymphocytes have shown that the MTHFR C677T polymorphism does not significantly affect uracil misincorporation and other markers of genomic instability 56, 57, although these negative observations might have resulted from supraphysiologic concentrations of riboflavin and methionine in culture medium 58. The effect of the MTHFR C677T polymorphism on uracil misincorporation and genomic instability in peripheral lymphocytes in human studies has been conflicting 50, 59-61. Again, these observations made in peripheral lymphocytes cannot be extrapolated to the colorectum or breast.

The observed effects of the MTHFR 677T mutation in the HCT116 and MDA-MB-435 cancer cells cannot be extrapolated to normal colon and breast epithelial cells, nor can they be generalized to other colon and breast cancer cell lines. Changes in intracellular methionine cycle intermediates and DNA methylation and enzymes regulating these processes in response to folate deficiency have previously been shown to be different between transformed and non-transformed epithelial cells and among transformed cell lines of the same cancer site 35, 42. Therefore, future studies investigating the functional effects of the MTHFR 677T variant in additional colon and breast cancer cell lines and in normal colon and breast epithelial cells are warranted to determine whether the differential effects observed in the present study are cancer-site specific differences or variability between cell lines. Mechanistic bases for the cell or tissue-specific functional effects of the MTHFR 677T variant, some of which are paradoxical, were not explored in the present study and hence, need to be clearly elucidated in future studies.

In conclusion, in HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect in the setting of folate insufficiency. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T variant in cells derived from the target organs of interest, thus providing proof of principle. Our data provide a possible mechanistic explanation for the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T variant observed in epidemiologic studies 7, 11, 12.

Acknowledgments

This research was supported by the Canadian Institutes of Health Research (Grant #14126 to YIK), the National Institutes of Health (R01 AG25834 and R21 AA16681 to SWC), and the National Science Council of Taiwan (Grant #NSC 95-2320-B-005-008-MY3 to EPC).

All authors have declared all sources of funding for research reported in this manuscript and have no financial or other contractual agreements that might cause conflicts of interest or be perceived as causing conflicts of interest. Dr. Peter Laird serves as a consultant and is on the scientific advisory board of Epigenomics, AG. However, this work was not supported by Epigenomics, AG.

Abbreviations

CpG
cytosine-guanine dinucleotide
DNMT
DNA methyltransferase
MTFHR
5,10-methylenetetrahydrofolate reductase
SAH
S-adenosylhomocysteine
SAM
S-adenosylmethionine
THF
tetrahydrofolate

Footnotes

Novelty and Impact: Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR C677T polymorphism in cells derived from the target organs of interest. Our data clearly demonstrate that the MTHFR C677T polymorphism affects the markers of folate metabolism, DNA synthesis and DNA methylation in a site-specific manner. Furthermore, our data show interactions between this polymorphism and exogenous folate levels in influencing DNA methylation. We provide biologically plausible mechanisms that may partly explain the epidemiologic observations that suggest that the MTHFR C677T polymorphism may modulate the risk of colorectal and breast cancers in a site-specific manner. The role of the MTHFR C677T polymorphism in cancer risk modification has major health implications given that it is a common mutation with an allele frequency of about 35% and that the target cancers are two of the most common cancers globally.

References

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