Epigenetic modifications on WNT genes by dietary genistein during colon cancer development

Epigenetic modifications, including DNA methylation and histone modifications switch the expression of genes on and off, and are crucial for normal development. Abnormal epigenetic events lead to carcinogenesis. Dietary factors have the ability to alter the epigenetic structure of gene and greatly contribute to the risk of colon cancer development. Epidemiological studies have shown that compared to the US, East Asia has a much lower incidence of colon cancer. One major difference in diet between these two populations is that East Asians consume more soy products. Genistein, a soy-derived isoflavone has been shown to act as an epigenome modifier, and to have anti-cancer properties. We aimed to explore the role(s) of soy isoflavone genistein during the development of colon cancer. Using a human colon cancer cell line DLD-1 as an in vitro experimental platform, we have identified that genistein restored the expression of a Wnt antagonist sFRP2, which was silenced in colon cancer cells, by affecting the DNA methylation at the promoter of the gene. Consequently, the abnormally activated Wnt signaling was attenuated, leading to repressed cancer cell viability and increased apoptosis. By using an azoxymethane (AOM)-induced rat colon cancer model, we examined the ability of genistein to modify the epigenetic structure of Wnt signaling genes, which have been shown to be closely involved in colon carcinogenesis. Male Sprague-Dawley rats were fed a control (CTL), a soy protein isolate (SPI), or a genistein (GEN) diet from gestation through 13-wk of age. The first sampling was conducted at 7 wk of age for pre-AOM analysis. The remaining rats were injected with AOM at 7-wk of age. Descending colon was collected 6 wk later for evaluation of aberrant crypt foci (ACF), gene expression, and nuclear protein accumulation. AOM injection induced aberrant nuclear accumulation of β-catenin in the CTL but not in the SPI or GEN groups. Moreover, WNT target genes Cyclin D1 and c-Myc were repressed by SPI and GEN. Meanwhile, SPI and GEN suppressed the expression of WNT signaling genes including Wnt5a, Sfrp1, Sfrp2, and Sfrp5 to the similar level to that of pre-AOM period. Rats fed SPI and GEN had a decreased number of total aberrant crypts. GEN feeding also resulted in a reduced number of ACF with N=3 per foci. The reduction of WNT/β-catenin signaling was correlated with the decrease of total aberrant crypts. By testing WNT/β-catenin signaling as a biomarker of colon carcinogenic potential, we showed the novel role of genistein as a suppressor of carcinogen-induced WNT/β-catenin signaling in preventing the development of early colon neoplasia. Next, we elucidated the epigenetic mechanisms by which genistein maintains a normal level of WNT genes during colon cancer development. We hypothesized that SPI and GEN induced epigenetic modifications on Sfrp2, Sfrp5, and Wnt5a genes, therefore suppressed their gene expression induced by AOM a level similar to that of the pre-AOM period. We identified that in the post-AOM period the acetylation of histone H3 (H3Ac) was downregulated by SPI and GEN at the promoter region of Sfrp2, Sfrp5, and Wnt5a, which paralleled the reduced binding of RNA Polymerase II. Nuclear level of histone deacetylase 3 (HDAC3) was enhanced by SPI and GEN. SPI and GEN suppressed the trimethylation of histone H3 Lysine 9 (H3K9Me3) and the phosphorylation of histone H3 Serine 10 (H3S10P). Methylation of the specific region of Sfrp2, Sfrp5, and Wnt5a genes was increased by SPI and GEN, which was inversely correlated with the reduced gene expression. Bisulfite sequencing further confirmed that dietary genistein induced DNA methylation at CpG island of the promoter region of Sfrp5. Importantly, this region includes a fragment that had decreased acetylation of histone H3. Our study presents a potential epigenetic mechanism by which dietary genistein controls the responses of WNT genes during carcinogen induction, which involves DNA methylation, histone modifications, and their interactions at the regulatory region of genes