Genistein suppresses colon cancer metastasis through epigenetic regulations

Colorectal cancer (CRC) is the third most common cancer in the United States. Approximately 90% of colon cancer deaths arise from cancer metastasis, the process by which primary tumors invade into and start to proliferate in a distant new site. Dietary factors are closely associated with development and progression of colon cancer. Genistein, one of the major phytochemicals in soy, has been shown to suppress metastasis in different cancer types including colon cancer. Moreover, studies have indicated that genistein is a mediator of epigenetic regulations, including DNA methylation and histone modifications, both in vitro and in vivo. But there is lack of critical data indicating the epigenetic mechanisms of metastasis prevention potentials of genistein. Because of the known functions of genistein in the suppression of cancer metastasis and its epigenome-modifying capabilities, we hypothesized that genistein can repress colon cancer metastasis, which involves epigenetic regulation. To test our hypothesis, first of all, the role of epigenetic regulation in colon cancer metastasis was investigated. Our research was focused on the study of metastasis suppressor, a type of molecules that inhibit cancer metastasis, in cell model SW480 and SW620. SW480 and SW620 are two colon cancer cell lines established from the same patient with different metastatic potentials, making them an ideal model for investigation of metastatic mechanisms. N-Myc downstream-regulated gene 1 (NDRG1) is known as a metastasis suppressor. We showed that NDRG1 mRNA in primary colon cancer cell line SW480 was forty times higher than that in highly metastatic SW620. Knockdown of NDRG1 in SW480 to a level that is similar to that in SW620 also modulated cell cycle and proliferation in SW480 towards the status of the highly metastatic SW620. Both DNA methylation and histone modification of NDRG1 in SW480 and SW620 were tested. We found that the silencing of NDRG1 in SW620 was not due to promoter hypermethylation. Chromatin immunoprecipitation revealed gene-wide decrease in histone H4 acetylation (H4Ac) and increase in phosphorylated histone H3 serine 10 (pH3S10) at the NDRG1 promoter in SW620. Meanwhile, the NDRG1 coding region showed much higher histone H3 lysine 4 methylation (H3K4me2) in SW480. These unique histone modifications in two colon cancer cell lines with different metastatic potentials suggested metastasis suppressor NDRG1 was epigenetically regulated in metastatic colon cancer cell line. In addition to NDRG1, dramatic down-regulation of Wnt5a, one of the WNT signaling factors, was also found in SW620 compared to SW480. Wnt5a expression was not responsive to DNA methyltransferase inhibitor 5-aza-cytidine treatment. However, histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate (NaBt) both significantly increased the mRNA expression of Wnt5a in SW620. NaBt treatment increased β-catenin signaling and diminished the difference in cell adhesion ability between SW480 and SW620, suggesting that the HDAC inhibitor plays critical roles in Wnt signaling pathway and cell physiology that relate to metastasis. Importantly, the lower transcription of Wnt5a in SW620 than SW480 corresponded to multiple histone modifications, including lower levels of acetylated histone H3 and H4 (H3Ac and H4Ac), dimethyl H3 lysine 4 (H3K4me2), and higher levels of trimethyl H3 lysine 27 (H3K27me3) in the promoter region. These results suggested the importance of Wnt5a in colon cancer metastasis and also indicated that the silencing of Wnt5a in the highly invasive human colon cancer cell line might result from transcriptional regulation of the gene by histone modifications. With the above findings, which indicated that epigenetic regulation of metastasis suppressor genes (specifically NDRG1 and Wnt5a) played an important role during colon cancer metastasis, next we tested the hypothesis that genistein represses colon cancer metastasis in vitro by modifying the expression of metastasis suppressor NDRG1 via epigenetic regulation. Genistein treatment extensively changed cellular properties of SW480 and SW620. For example, Genistein reduced the proliferation and migration of both cell lines. Genistein specifically increased the adhesion of the metastatic cell line SW620, which was correlated to increased expression of E-cadherin and the decrease of matrix metalloproteinases-7 (MMP-7). Moreover, genistein increased the mRNA expression of NDRG1. The enhanced NDRG1 gene expression in SW620 was associated with increased gene transcription activity and altered histone structure of the gene. H3K36me3, a positive histone modification, was increased by genistein at 5’untranslated region (5’UTR) and distal downstream regions. Genistein also caused global decrease of H3S10p in both cell lines. The following study aimed at investigating the effect of genistein on liver metastases derived from colon cancer and the molecular mechanism behind in an in vivo model. Male athymic nude mice were fed with western diet (W; calories%, fat 39%, carb 50%), W diet supplemented with 100ppm Gen (GL) or 500ppm Gen (GH) . After 16 weeks, all the animals were intrasplenically injected with metastatic human colon cancer cells SW620 and sacrificed 8 weeks later. GL and GH mice showed lower % liver metastases (50% and 27.3%) compared to W group (80% of mice with metastases). Pathological evaluations of representative liver sections from all animals showed no significant differences of liver metastases derived from injected SW620, such as metastases size, % of necrosis and mitotic index. mRNA expressions of metastasis-related human genes tested by real-time PCR also showed no difference of gene expression. Only the proliferation index represented by pH3S10 positive cells in liver metastases in W group was higher than GL and GH groups. Intriguingly, the expression of both β-catenin (key intracellular mediator of the Wnt signaling pathway) and NDRG1 were increased by dietary genistein in normal liver sections, suggesting the liver microenvironment was altered by genistein, which might be highly responsible for the lower metastasis incidence in liver in genistein treatment group. The results of this study for the first time demonstrated the anti-metastatic function of genistein in colon cancer metastasis both in vitro and in vivo. Epigenetic regulation, especially histone modification, was shown to play an important role in colon cancer metastasis through regulating the expression of metastasis suppressor genes in our study. The findings from our study increase the understanding of the anti-metastatic effect of genistein in colon cancer metastasis and provide important information for applying genistein in therapy to prevent metastasis