Intestinal PPARα Protects Against Colon Carcinogenesis via Regulation of Methyltransferases DNMT1 and PRMT6

Abstract

Background & aims: Many genetic and environmental factors, including family history, dietary fat, and inflammation, increase risk for colon cancer development. Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor that regulates systemic lipid homeostasis. We explored the role of intestinal PPARα in colon carcinogenesis.

Methods: Colon cancer was induced in mice with intestine-specific disruption of Ppara (Ppara^ΔIE), Ppara^fl/fl (control), and mice with disruption of Ppara that express human PPARA (human PPARA transgenic mice), by administration of azoxymethane with or without dextran sulfate sodium (DSS). Colons were collected from mice and analyzed by immunoblots, quantitative polymerase chain reaction, and histopathology. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses were performed on urine and colons. We used molecular biology and biochemical approaches to study mechanisms in mouse colons, primary intestinal epithelial cells, and colon cancer cell lines. Gene expression data and clinical features of patients with colorectal tumors were obtained from Oncomine, and human colorectal-tumor specimens and adjacent normal tissues were collected and analyzed by immunohistochemistry.

Results: Levels of Ppara messenger RNA were reduced in colon tumors from mice. Ppara^ΔIE mice developed more and larger colon tumors than control mice following administration of azoxymethane, with or without DSS. Metabolomic analyses revealed increases in methylation-related metabolites in urine and colons from Ppara^ΔIE mice, compared with control mice, following administration of azoxymethane, with or without DSS. Levels of DNA methyltransferase 1 (DNMT1) and protein arginine methyltransferase 6 (PRMT6) were increased in colon tumors from Ppara^ΔIE mice, compared with colon tumors from control mice. Depletion of PPARα reduced the expression of retinoblastoma protein, resulting in increased expression of DNMT1 and PRMT6. DNMT1 and PRMT6 decreased expression of the tumor suppressor genes Cdkn1a (P21) and Cdkn1b (p27) via DNA methylation and histone H3R2 dimethylation-mediated repression of transcription, respectively. Fenofibrate protected human PPARA transgenic mice from azoxymethane and DSS-induced colon cancer. Human colon adenocarcinoma specimens had lower levels of PPARA and retinoblastoma protein and higher levels of DNMT1 and PRMT6 than normal colon tissues.

Conclusions: Loss of PPARα from the intestine promotes colon carcinogenesis by increasing DNMT1-mediated methylation of P21 and PRMT6-mediated methylation of p27 in mice. Human colorectal tumors have lower levels of PPARA messenger RNA and protein than nontumor tissues. Agents that activate PPARα might be developed for chemoprevention or treatment of colon cancer.

Keywords: Epigenetic; Mouse Model; Transcriptional Regulation.

Figure 1.. The expression of PPARα was decreased in human and mouse colon tumors.

(A) Analysis of PPARA gene expression from the online TCGA Colorectal 2 dataset and (B) differential PPARA gene expression based on disease stages. Analysis of PPARA (C) and ACOX1 (D) gene expression from the online Ki colon dataset. (E) Representative IHC staining of PPARα on human normal mucosa and adenocarcinoma. Scale bars: upper, 300 μm; bottom, 200 μm. (F) The IHC staining intensity of PPARα on human colon specimens. (G) Ppara and Acoxl mRNA levels and (H) PPARα and ACOX1 protein levels in mouse colon tumors (T) and adjacent non-tumor tissues (NT). N = 5/group, for mRNA analysis. * P< 0.05, ** P< 0.01, *** P< 0.001.

Figure 2.. Intestine-specific knockout of PPARα increased AOM and AOM and DSS induced colon carcinogenesis.

For AOM model, (A) Representative gross pictures (upper) and H&E staining (bottom) of colon sections. Scale bars: upper, 1.5 mm; bottom, 100 μm. (B) Prevalence of low-grade adenomas, high-grade adenomas and adenocarcinomas. (C) Colon length. (D) Tumor number. (E) Number of tumors with diameters > 3 mm. (F) Number of tumors with diameters < 3mm. For AOM and DSS model, (G) Colon length. (H) Tumor number. (I) Prevalence of low-grade adenomas, high-grade adenomas and adenocarcinomas. (J) Representative gross pictures (upper) and H&E staining (bottom) of colon sections. Scale bars: upper, 1.5 mm; bottom, 100 μm. * P< 0.05.

Figure 3.. Several methylation-related metabolites were increased in the colon of tumor-bearing Ppara^ΔIE mice.

(A) Score scatter plot for principal components analysis of data obtained from HILIC analysis of colon samples from AOM mouse model. N=6 mice/group. (B) Relative levels of indicated metabolites in the colon samples described in (A). (C) Score scatter plot for principal components analysis of data obtained from HILIC analysis of colon samples from AOM and DSS mouse model. N=3–6 mice/group. (D) The relative levels of indicated metabolites in the colon samples described in (C). * P< 0.05, ** P< 0.01, *** P< 0.001.

Figure 4.. Intestine-specific knockout of PPARα increased the expression of DNMT1 and PRMT6.

(A) The mRNA levels of methyltransferases in mouse colon tumors (T) and adjacent non-tumor tissues (NT). N = 5/group. (B) The mRNA levels of methyltransferases in the colon samples from Ppara^fl/fl and Ppara^ΔIE mice. N=5/group. (C) Western blot analysis of indicated proteins in mouse colon tumors (T) and adjacent non-tumor tissues (NT). (D) Western blot analysis of indicated proteins in colon samples from Ppara^fl/fl and Ppara^ΔIE mice. (E) The mRNA levels of Ppara, Acox1, Dnmt1, and Prmt6 in colon tumors (T) and adjacent non-tumor tissues (NT) from AOM-administered Ppara^fl/fl and Ppara^ΔIE mice (n=5/group). (F) The mRNA (n=3) and (G) protein levels of DNMT1 and PRMT6 in primary intestinal epithelial cells isolated from Ppara^fl/fl and Ppara^ΔIE mice. (H) Primary intestinal epithelial cells isolated from wild-type mice were cultured and infected with control adenovirus (Ad-GFP), adenovirus expressing PPARα (Ad-PPARα), or not infected (No-Ad). The mRNA (n=2 for No-Ad group, and n=4 for Ad-GFP group and Ad-PPARα group) and (I) protein levels of indicated genes were analyzed. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 5.. DNMT1 and PRMT6 suppressed the expression of p21 and p27 by DNA methylation and histone arginine methylation, respectively.

(A) The mRNA levels of tumorigenesis related genes in mouse colon tumors (T) and adjacent non-tumor tissues (NT). N = 5/group. (B) The mRNA levels of tumorigenesis related genes in the colon samples from Ppara^fl/fl and Ppara^ΔIE mice. N=5/group. (C) The mRNA (n=4/group) and (D) protein levels of indicated genes in HCT116 cells transfected with empty vector pcDNA3 or plasmid expressing DNMT1 (pDNMT1), or exposed to DMSO (Ctrl) or 5-Aza. (E) The mRNA (n=4/group) and (F) protein levels of indicated genes in HCT116 cells transfected with empty vector pCMV or plasmid expressing PRMT6 (pPRMT6), or administered with DMSO (Ctrl) or EPZ020411 (EPZ). (G) Genomic DNA was isolated from HCT116 cells described in (D) and subjected to MeDIP analysis (n=3/group). (H) Genomic DNA was isolated from colon tissues of mice described in Figure 3A and subjected to MeDIP analysis (n=3/group). Values were calculated as percentages of input DNA and expressed as relative enrichment compared to control group, which was equated to 1. (I) Histone ChIP assay was performed using chromatins prepared from HCT116 cells described in (F) (n=3/group). (J) Histone ChIP assay was performed using chromatins prepared from colon tissues of mice described in Figure 3A (n=3/group). Values were expressed as relative enrichment compared to histone H3. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 6.. Intestinal PPARα regulated the expression of DNMT1 and PRMT6 via RB1/E2F pathway.

(A) The mRNA (n=4/group) and (B) protein levels of indicated genes in primary intestinal epithelial cells exposed to DMSO (Ctrl) or PD0332991. (C) Schematic diagram of the mouse Rb1 promotor illustrating the potential PPREs in the regulatory region and the fragments used for luciferase reporter assay. The upstream regions were numbered in relation to the transcription initiation site, which was designated +1. (D) Luciferase reporter assay of mouse Rb1 promoter activity (n=3/group). ChIP assays on chromatins isolated from MC38 cells for Rb1 (E), Acoxl (F), and Acotl (G). N=3/group. * P< 0.05, ** P< 0.01, *** P< 0.001.

Figure 7.. Fenofibrate decreased AOM and DSS-induced colon carcinogenesis in human PPARA transgenic mice.

(A) Colon length. (B) Tumor number. (C) Prevalence of low-grade adenomas, high-grade adenomas and adenocarcinomas. (D) Representative gross pictures (upper) and H&E staining (bottom) of colon sections. Scale bars: upper, 1.5 mm; bottom, 100 μm. (E) Left, Representative BrdU staining of colon sections. Scale bars: 50 μm. Right, BrdU labeling index. (F) The mRNA (n=8) and (G) protein levels of indicated genes in the colon samples of AOM and DSS-administered human PPARA transgenic mice fed on chow diet or fenofibrate diet. * P< 0.05, ** P< 0.01, *** P< 0.001.