Altered folate availability modifies the molecular environment of the human colorectum: implications for colorectal carcinogenesis

Abstract

Low folate status increases colorectal cancer risk. Paradoxically, overly abundant folate supplementation, which is not uncommon in the United States, may increase risk. The mechanisms of these effects are unknown. We conducted two translational studies to define molecular pathways in the human colon altered either by folate supplementation or by dietary folate depletion (followed by repletion). In the first study, 10 healthy, at-risk volunteers (with documented stable/normal folate intake) received supplemental folic acid (1 mg/d) for 8 weeks. In the second study, 10 similar subjects were admitted to a hospital as inpatients for 12 weeks to study folate depletion induced by a low folate diet. A repletion regimen of folic acid (1 mg/d) was provided for the last 4 of these weeks. Both studies included an 8-week run-in period to ensure stabilized folate levels prior to intervention. We obtained 12 rectosigmoid biopsies (from 4 quadrants of normal-appearing mucosa 10-15 cm from the anal verge) at baseline and at measured intervals in both studies for assessing the primary endpoints: genome-wide gene expression, genomic DNA methylation, promoter methylation (depletion/repletion study only), and p53 DNA strand breaks. Serum and rectosigmoid folate concentrations accurately tracked all changes in folate delivery (P < 0.05). In the first study, gene array analysis revealed that supplementation upregulated multiple inflammation- and immune-related pathways in addition to altering several 1-carbon-related enzymes (P < 0.001). In the second study, folate depletion downregulated genes involved in immune response, inflammation, the cell cycle, and mitochondrial/energy pathways; repletion reversed most of these changes. However, changes in gene expression after repletion in the second study (involving immune response and inflammation) did not reach the levels seen after supplementation in the first study. Neither genomic nor promoter-specific DNA methylation changed during the course of the depletion/repletion protocol, and genomic methylation did not change with supplementation in the first study. p53 DNA strand breaks increased with depletion after 12 weeks. In sum, depletion downregulates, whereas repletion or supplementation upregulates pathways related to inflammation and immune response. These findings provide novel support to the concept that excessive folate supplementation might promote colorectal carcinogenesis by enhancing proinflammatory and immune response pathways. These results indicate that modest changes in folate delivery create substantial changes in the molecular milieu of the human colon.

Trial registration: ClinicalTrials.gov NCT00220012.

©2011 AACR.

Figures

Figure 1

Figure shows a diagram of the 2 parallel folate intervention trials. Note that run-in period was identical for the two studies. Time-points of rectosigmoid biopsies collections are shown in the diagram; at these time-points blood endpoints were also measured.

Figure 2

Changes in serum, rectosigmoid, and RBC folate concentrations and serum homocysteine concentrations after dietary folate depletion followed by repletion and after folic acid supplementation. Data is shown as individual data points for all 10 subjects. P values are shown in the figure, horizontal lines indicate mean values. Parallel changes in serum and rectosigmoid folate concentrations occurred as a result of depletion and supplementation and were accompanied by reciprocal changes in serum homocysteine, validating the biochemical effects of folate depletion, repletion, and supplementation.

Figure 3

Intracellular 1-carbon metabolism (simplified), emphasizing pathways relevant to the text. Multiple significant alterations in gene expression in this metabolic pathway were observed. Enzyme gene names are shown in capital letters, upregulated genes are shown in green and downregulated genes red, genes whose expression did not change are shown in black. Major end-products relevant to the discussion are in bold italics. Methionine adenosyltransferase 2b (MAT2b) is upregulated but all other genes whose expression were altered were downregulated. Notably thymidylate synthase (TYMS),dihydrofolate reductase (DHFR) and 2 de novo DNA methyltransferases 3A and 3B (DNMT3A and DNMT3B) are downregulated. CTH (cystathionine lyase) is reduced as well. The function of the gene product of AHCYL1 (S-adenosylhomocysteine hydrolase-like 1) is not known with certainty but a highly homologous protein catalyzes the reversible hydrolysis of S-adenosylhomocysteine (S-Adohcy) to adenosine and L-homocysteine. Additional genes that were downregulated include folate receptor 1 (FOLR1); gamma glutamyl hydrolase (‘conjugase’,GGH), an enzyme that cleaves the polyglutamyl tail of folates; methylenetetrahydrofolate dehydrogenease (MTHFD1), which is involved in the interconversion of 1-carbon derivatives of tetrahydrofolate; 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), which regenerates an active form of methionine synthase (MTR) via reductive methylation with vitamin B12 as cofactor; and phosphoribosylglycinamide formyltransferase (GART), which is pivotal in purine biosynthesis. THF = tetrahydrofolate; S-AdoMet = S-adenosylmethionine; S-AdoHcy = S-adenosylhomocysteine.

Figure 4

Heatmap of Gene Set Enrichment Analysis for Gene Ontology across folate depletion-repletion study and folic acid supplementation study. Only categories with FDR-q-value of less than 0.001 in at least one condition are shown in the figure. Colors indicate down-regulation (green) or up-regulation (red) and values are 1 - P values (with down-regulation given negative values). DR indicate depletion-repletion study and S indicates folate supplementation study. Figure includes enrichment results for comparison of nadir of depletion (8 weeks) vs 4 week repletion timepoints. Note that categories related to immune functions and inflammation are strongly associated with folate status. To demonstrate these changes on individual gene level, lower part of the figure shows general relative changes of significantly expressed genes that belong to immune response process and immune response GOs and whose expression is linked to folate status, left part for depletion-repletion and right panel for supplementation study. Individual contributing genes are listed in the Supplement S4.

Figure 5

Changes in gene expression in rectosigmoid mucosa by quantitative RT-PCR. Gene symbols are listed on a top of each graph. Data is shown as individual paired expression data points for all 8 subjects at baseline and after 8 weeks of folate supplementation. To validate the arrays three most upregulated genes (LTF, CCL20 and TLC1) and the 3 most downregulated genes (BEST4, GCG and PYY) were measured. To validate the changes in inflammatory genes and genes involved in 1-carbon metabolism OLFM4, IL-6, DHFR and TYMS were also measured. P values are listed in the graph.