Loss of FFAR2 promotes colon cancer by epigenetic dysregulation of inflammation suppressors

Abstract

Free fatty acid receptor 2 (FFAR2, also named GPR43), is activated by short-chain fatty acids (SCFAs), such as butyrate, that are produced when gut bacteria ferment dietary fiber. FFAR2 has been suggested to regulate colonic inflammation, which is a major risk factor for the development of colon cancer and is also linked to epigenetic dysregulation in colon carcinogenesis. The current study assessed whether FFAR2, acting as an epigenetic regulator, protects against colon carcinogenesis. To mimic the mild inflammation that promotes human colon cancer, we treated mice with dextran sodium sulfate (DSS) overnight, which avoids excessive inflammation but induces mild inflammation that promotes colon carcinogenesis in the Apc^Min/+ and the azoxymethane (AOM)-treated mice. Our results showed that FFAR2 deficiency promotes the development of colon adenoma in the Apc^Min/+ /DSS mice and the progression of adenoma to adenocarcinoma in the AOM/DSS mice. FFAR2's downstream cAMP-PKA-CREB pathway was enhanced, leading to overexpression of histone deacetylases (HDACs) in the FFAR2-deficient mice. ChIP-qPCR analysis revealed differential binding of H3K27me3 and H3K4me3 histone marks onto the promoter regions of inflammation suppressors (e.g., sfrp1, dkk3, socs1), resulting in decreased expression of these genes in the FFAR2-deficient mice. Also, more neutrophils infiltrated into tumors and colon lamina propria of the FFAR2-deficient mice. Depletion of neutrophils blocked the progression of colon tumors. In addition, FFAR2 is required for butyrate to suppress HDAC expression and hypermethylation of inflammation suppressors. Therefore, our results suggest that FFAR2 is an epigenetic tumor suppressor that acts at multiple stages of colon carcinogenesis.

Keywords: FFAR2; HDAC; butyrate; colon cancer; epigenetics.

Figure 1. FFAR2 deficiency promoted the development of adenomas and the progression of adenoma to adenocarcinoma, and enhanced the downstream cAMP–PKA–CREB–HDAC pathway

(A) The Apc^Min/+ (n=15) and Apc^Min/+-FFAR2^−/− (n=15) mice were treated with 5% DSS overnight and were euthanized 6 weeks after the DSS treatment. (B) The Apc^Min/+-FFAR2^−/−/DSS mice developed more and larger lesions that showed high-grade dysplastic features. (C) Staining of cAMP–PKA–CREB–HDAC signaling, proliferative marker (Ki67) and GR-1 neutrophil in colon adenoma was enhanced in colon of the Apc^Min/+-FFAR2^−/−/DSS mice (Apc^Min/+: n=4–5, Apc^Min/+-FFAR2^−/−: n=4–5). (D) Wild-type (WT) (n=15) and FFAR2^−/− (n=15) mice were treated with one dose of AOM (15mg/kg body weight, i.p.) followed by 5% DSS overnight. Six weeks after receiving DSS, all the mice were euthanized. (E) The FFAR2^−/− mice had more and larger tumors and a larger percentage of intramucosal adenocarcinoma. (F) Staining of cAMP–PKA–CREB–HDAC signaling, proliferative marker (Ki67) and GR-1 neutrophil in colon adenoma and adenocarcinoma was enhanced in colon of the AOM/DSS-treated FFAR2^−/− mice (WT: n=3, FFAR2^−/−: n=3). LGD: low-grade dysplasia; HGD: high-grade dysplasia: IMC: intramucosal adenocarcinoma. * p <0.05.

Figure 2. FFAR2 deficiency disrupted neutrophil homeostasis in colonic lamina propria

(A) The Apc^Min/+ and Apc^Min/+-FFAR2^−/− mice were treated with 5% DSS overnight and were euthanized 6 weeks after the DSS treatment. (B) Percentages of colonic LP neutrophils and cytokines were measured by flow cytometry (Apc^Min/+: n=16, Apc^Min/+-FFAR2^−/−: n=6). (C) Two weeks after the DSS treatment, Apc^Min/+-FFAR2^−/− mice were given anti-GR-1 antibodies (GR-1 Ab) once per week for 4 weeks. (D) Depletion of neutrophils by GR-1 Ab suppressed colon lesions in the Apc^Min/+-FFAR2^−/−/DSS mice (Ctrl: n=15, GR-1 Ab: n=5). (E) The wild-type (WT) and FFAR2^−/− mice were treated with one dose of AOM (15mg/kg body weight, i.p.) followed by 5% DSS overnight, and were euthanized 6 weeks after the DSS treatment. (F) Percentages of colonic LP neutrophils and cytokines were measured by flow cytometry (WT: n=7, FFAR2^−/−: n=4). * p <0.05.

Figure 3. FFAR2 deficiency changed histone modification on inflammation regulators

(A) The Apc^Min/+ and Apc^Min/+-FFAR2^−/− mice were treated with 5% DSS overnight and were euthanized 6 weeks after the DSS treatment. ChIP-qPCR analysis of H3K27me3 (B) and H3K4me3 (C) on the promoter regions of inflammation suppressors (triple technique repeats were shown), and mRNA expression of those genes (D) from colonic LP of the Apc^Min/+/DSS and Apc^Min/+-FFAR2^−/−/DSS mice. (E) The wild-type (WT) and FFAR2^−/− mice were treated with one dose of AOM (15mg/kg body weight, i.p.) followed by 5% DSS overnight, and were euthanized 6 weeks after the DSS treatment. ChIP-qPCR analysis of H3K27me3 (F) and H3K4me3 (G) on the promoter regions of inflammation suppressors (triple technique repeats were shown), and mRNA expression of those genes (H) from colonic LP of the AOM/DSS-treated WT and FFAR2^−/− mice (n=3). * p <0.05.

Figure 4. FFAR2 was required for butyrate to function in colonic epithelium and colonic LP

(A) The wild-type (WT) and FFAR2^−/− mice were treated with one dose of AOM (15 mg/kg body weight, i.p.) followed by 5% DSS overnight, and were euthanized two weeks after the DSS treatment. Colonic epithelium and LP were isolated from these mice and cultured with or without 5 mM butyrate for 16 hr. mRNA expression of hdac2, sox17, and socs1 and promoter DNA methylation of sox17 and socs1 in colonic epithelium (B) and colonic LP (C) (WT: n=6, FFAR2^−/−: n=6). (D) Percentage of neutrophils and IL-1β levels in colonic LP were measured by flow cytometry (WT: n=7–8, FFAR2^−/−: n=4). * p <0.05.

Figure 5. Loss of FFAR2 epigenetically promotes colon cancer

SCFAs bind to and activate FFAR2, which inhibits its downstream cAMP–PKA–CREB–HDAC pathway. SCFAs also can be transported across cell membrane to inhibit HDAC activities. Loss of FFAR2 results in enhanced cAMP–PKA–CREB pathway and overexpressed HDAC. The expression levels of inflammation suppressors, such as sfrp1, dkk3, and socs1, were decreased, which promote the progression of colon carcinogenesis. However, absorption-mediated HDAC inhibition by SCFAs could still occur in FFAR2-deficient cells.