Dietary folate deficiency blocks prostate cancer progression in the TRAMP model

Abstract

Dietary folate is essential in all tissues to maintain several metabolite pools and cellular proliferation. Prostate cells, due to specific metabolic characteristics, have increased folate demand to support proliferation and prevent genetic and epigenetic damage. Although several studies have found that dietary folate interventions can affect colon cancer biology in rodent models, its impact on prostate is unknown. The purpose of this study was to determine whether dietary folate manipulation, possibly being of primary importance for prostate epithelial cell metabolism, could significantly affect prostate cancer progression. Strikingly, mild dietary folate depletion arrested prostate cancer progression in 25 of 26 transgenic adenoma of the mouse prostate (TRAMP) mice, in which tumorigenesis is prostate-specific and characteristically aggressive. The significant effect on prostate cancer growth was characterized by size, grade, proliferation, and apoptosis analyses. Folate supplementation had a mild, nonsignificant, beneficial effect on grade. In addition, characterization of folate pools (correlated with serum), metabolite pools (polyamines and nucleotides), genetic and epigenetic damage, and expression of key biosynthetic enzymes in prostate tissue revealed interesting correlations with tumor progression. These findings indicate that prostate cancer is highly sensitive to folate manipulation and suggest that antifolates, paired with current therapeutic strategies, might significantly improve treatment of prostate cancer, the most commonly diagnosed cancer in American men.

Figure 1. Folate, one-carbon metabolism, methionine cycle and polyamine biosynthesis overview

Dietary folate (left) is necessary for de novo synthesis of deoxythymidine monophosphate (dTMP) and adenosylmethionine (AdoMet), which in turn are necessary for DNA synthesis and intracellular methylation reactions, respectively. AdoMet is also used for polyamine biosynthesis (top), which is prominent in prostate cells. dTMP can also be salvaged as thymidine from DNA degradation. The key enzyme responsible for de novo synthesis of dTMP is thymidylate kinase (TS), while the one for conversion of thymidine into dTMP is thymidine kinase (TK).

Figure 2. Dietary folate depletion prevents CaP development in the TRAMP model

(A) Pearson correlation analysis between serum and tissue folate levels as quantified by Lactobacillus Casei assay at 22 weeks (mice on all three diets are shown). A similar correlation was observed at 12 weeks (not shown). Liver (purple circles) and prostate (orange squares) folate levels from mice on the three folate defined diets are represented on the Y axis, while serum level of folate in the same animals is represented on the X axis. (B) Prostate tumor burden analysis. The ratio between the weight of the urogenital tract (UG) and the whole body of each mouse is represented on the Y axis according to the three folate defined diets. (C) Histopathological analysis of prostate tumors. Disease index, calculated as ( (average predominant H&E grade for each lobe+ average worst grade for each lobe) * UG weight), is represented on the Y axis for the cohorts of TRAMP mice.

Figure 3. Representative images of H&E staining on 22 weeks old TRAMP mice fed the folate defined diets as indicated

Dorsal (DP), lateral (LP), and ventral (VP) prostate lobes are shown from the same mouse per each diet.

Figure 4. Dietary folate depletion arrests CaP progression in the TRAMP model

Tabulation of immunohistochemical analysis of 22 weeks old TRAMP mouse ventral prostate (A) Hematoxylin and Eosin to assess tumor grading (H&E). Statistical analyses tested the difference in cases with a grade >3. (B) KI-67, to assess cellular proliferation. Statistical analyses tested the difference in cases with >25% positive cells. (C) Cleaved caspase 3, to assess apoptosis. Statistical analyses tested the difference in cases with >25% positive cells. (D) E-cadherin to assess differentiation. Statistical analyses tested the difference in cases with normal versus abnormal (weak or absent) staining. (E) Transgene staining (SV-40 large T antigen) of the lymph node to assess presence or absence of lymph node metastasis. Statistical analyses tested the difference in cases that were positive or negative for lymph node metastases. All statistical comparisons were made between the control diet and each of the experimental diets individually using a two-tailed Fisher’s Exact test.

Figure 5. Genetic and epigenetic damage assessment in TRAMP prostate

A) Mass array quantitative methylation analysis of four loci frequently methylated during tumor progression in the TRAMP model. The average level of methylation detected by MAQMA across the sequenced fragment of the CpG islands for each samples is shown on the Y axis. This value comes from taking the average of MAQMA values for each CpG dinucleotide sequenced for each sample. Each symbol represents a single sample. B) HPLC analysis of dTTP, dUTP, and the dUTP: dTTP ratio in 22 weeks old TRAMP mice fed the folate defined diets. C) Random oligo-primed synthesis (ROPS) analysis in 22 weeks old TRAMP prostate on the folate restricted diets quantifies DNA single strand breaks before and after the removal of misincorporated uracil. Single strand DNA breaks are directly proportional to the amount of radioactive dCTP incorporated (Y axis). Statistical comparisons were made between the control diet and each of the experimental diets individually using a two-tailed Mann Whitney test. Statistical significance is indicated as *=P<0.05, **=P<0.01, ***=P<0.001.

Figure 6. Expression of biosynthetic and salvage pathway enzymes for dTTP under the influence of dietary folate manipulation

(A) Correlation of TS (left) and TK (right) expression with the UG/body weight at 22 weeks, regardless of the diet. (B) Real-time RT PCR analysis of TS ( top) and TK (bottom) expression normalized to 18s in 22 week old TRAMP prostate categorized by the folate defined diets. The full dot represents data from the one animal that developed a large tumor on the folate deficient diet.