Dietary folate levels alter the kinetics and molecular mechanism of prostate cancer recurrence in the CWR22 model

Abstract

Folate impacts the genome and epigenome by feeding into one-carbon metabolism to produce critical metabolites, deoxythymidine monophosphate and s-adenosylmethionine. The impact of folate exposure and intervention timing on cancer progression remains controversial. Due to polyamine metabolism's extraordinary biosynthetic flux in prostate cancer (CaP) we demonstrated androgen stimulated CaP is susceptible to dietary folate deficiency. We hypothesized dietary folate levels may also affect castration recurrent CaP. We used the CWR22 human xenograft model which recurs following androgen withdrawal. Engrafted mice were fed a folate depleted or supplemented diet beginning at androgen withdrawal, or prior to xenograft implantation. Both folate depletion and supplementation at the time of withdrawal significantly decreased recurrence incidence. Folate supplementation prior to xenograft implantation increased time to recurrence, suggesting a protective role. By contrast, folate depleted recurrent tumors exhibited transcriptional adaptive responses that maintained high polyamine levels at the expense of increased DNA damage and DNA methylation alterations. Mining of publically available data demonstrated folate related pathways are exceptionally dysregulated in human CaP, which correlated with decreased time to biochemical recurrence. These findings highlight the potential for novel therapeutic interventions that target these metabolic pathways in CaP and provide a rationale to apply such strategies alongside androgen withdrawal.

Keywords: androgen withdrawal; castration recurrent prostate cancer; folate; one-carbon metabolism; polyamine metabolism.

Figure 1. Overview of one-carbon metabolism, polyamine biosynthesis, and the methionine salvage pathway

Key enzymes are italicized and underlined. TYMS, thymidylate synthase; TK1, thymidine kinase; MTHFR, methylenetetrahydrofolate reductase; AMD1, s-adenosylmethionine decarboxylase; ODC1, ornithine decarboxylase; SRM, spermidine synthase; SMS, spermine synthase; SAT1, spermidine/spermine N1-acetyltransferase; MTAP, methylthioadenosine phosphorylase; SLC3A2, solute carrier family 3 member 2.

Figure 2. Dietary folate levels alter the kinetics of prostate cancer recurrence

(A) Experimental study design. In study 1 diets were started at the time of androgen withdrawal. In study 2 diets were started ∼14 days prior to xenograft injection. Tumors were followed for a maximum of 50 weeks post androgen withdrawal. 4 points of observation were noted, initial response (defined as reaching < 300 mm³ (the original tumor volume at androgen withdrawal), regression (< 150 mm³), complete regression (< 100 mm³) and recurrence (> 500 mm³). (B–D) Analysis of recurrence status every 4 weeks post androgen withdrawal. The graph indicates the percent of animals every 4 weeks considered to have complete regression (black circle), stable disease (dark grey square) and recurrence (grey triangle). (E) Percent cases which recurred for each diet in study 1, and depleted and supplemented diets from study 2 by week 42. (F) Number of animals that completely regressed (reached < 100 mm³) in animals on a supplemented diet at androgen withdrawal and supplementation prior to xenograft implantation. (G) Week of recurrence for animals on a supplemented diet in study 1 and study 2. AW refers to androgen withdrawal. Statistical analyses were made using a Fisher’s Exact test comparing number of recurred and non-recurred cases. (^*p < 0.05)

Figure 3. Polyamine biosynthesis is altered in response to dietary manipulation

(A) Total polyamine levels (putrescine + spermidine + spermine) normalized to milligram of protein as measured by UPLC for recurrent and stable disease tumors in each diet from study 1. (B) S-adenosylmethionine decarboxylase (AMD1) expression normalized to glucuronidase beta (GUSB) as measured by Real Time RT-PCR for recurrent and stable disease tumors in each diet from study 1. (C) The SAM to SAH ratio as measured by UPLC for recurrent tumors in each diet from study 1. (D) Solute carrier family 3 member 2 (SLC3A2) mRNA expression for recurrent tumors in study 1. (E) Polyamine levels for animals on the supplemented diets in study 1 and study 2. (F) Pearsons’ correlation between polyamine levels and week of recurrence in study 2. AW refers to androgen withdrawal. Statistical analyses were made using an unpaired student t-test with Welch’s correction. Correlations were calculated within each diet by 2-tailed Pearson correlation test. (^***p < 0.001)

Figure 4. Folate uptake and retention enzymes are altered in response to folate depletion

(A) mRNA expression levels as measured by Real Time RT-PCR for the reduced folate carrier (RFC) and (B) folylpolyglutamate synthase (FPGS) normalized to glucuronidase beta (GUSB) in the recurrent and stable disease tumors for each diet from study 1. AW refers to androgen withdrawal. Statistical analyses were made using an unpaired student t-test with Welch’s correction (^*p < 0.05: ^***p < 0.001)

Figure 5. One-carbon metabolism is altered in response to folate depletion

(A) mRNA expression levels normalized to GUSB as measured by Real Time RT-PCR for thymidine kinase (TK1) in recurrent and stable disease tumors for each diet in study 1. (B) Thymidylate synthase (TYMS) mRNA expression in recurrent tumors for each diet in study 1. (C) Correlation of TYMS and TK1 for recurrent tumors in each diet in study 1. (D) deoxyuridine monophosphate (dUMP) levels as measured by HPLC normalized to milligrams of tissue for recurrent tumors in study 1. (E) Quantification by densitometry of Western blot analysis of γH2AX protein expression normalized to β-Actin for recurrent tumors in study 1. Representative images for blots are presented for 10 animals from each diet. (F) Correlation of dUMP with TK1, (G) TYMS and (H) γH2AX. Statistical analyses were performed for average expression values of each gene or metabolite levels using an unpaired student t-test with Welch’s correction. Correlations were calculated by 2-tailed Pearson correlation test (^*p < 0.05: ^**p < 0.01: ^***p < 0.001).

Figure 6. DNA Methylation is altered in response to dietary interventions

(A) Number of CpG sites with > 30% increase in methylation (hypermethylation) or > 30% decrease in methylation (hypomethylation), relative to the average methylation observed in the 4 control animals, in at least two of the four animals tested for each diet in study 1 recurrent tumors. (B) Venn Diagram indicating a significant overlap between genes associated with differential DNA methylation from animals on the depleted diet, and genes differentially methylated in at least 2 studies utilizing the TCGA-PRAD cohort. Statistical analysis was performed using a hypergeometric distribution. (C) Networks for the top 20 enriched gene sets from the hypomethylated genes and (D) hypermethylated genes from depleted recurrent tumors. Node size is indicative of the log(p-value) for enrichment. Degree of shading of nodes indicates percentage of methylated genes found within the gene set. Edge width is indicative of the degree of overlap between gene sets.

Figure 7. Human Data reveals dysregulation of folate, one-carbon and polyamine metabolism in prostate cancer

(A) Heatmap representing clustering of tumor samples based on mRNA expression of a panel of 13 candidate genes normalized to GUSB expression relative to median expression of each gene. Color coding in left-most column indicates the diet group the tumor came from and the tumor status as recurrent or stable disease. (B) Absolute value scores for the degree of dysregulation of genes within the Folate Metabolism, Folate One-Carbon Metabolism and Polyamine Biosynthetic Pathways for the indicated number of cases in six TCGA cohorts. (C) Five-year biochemical recurrence (BCR) free survival curves based on lower (Q1 = Quartile 1) and upper (Q4 = Quartile 4) quartiles of dysregulation in Folate Metabolism, (D) Folate One-Carbon Metabolism and (E) Polyamine Metabolism pathways. Statistical analyses are detailed in methods section.