Abstract 2788: Inhibition of DNA methylation suppresses intestinal tumor organoids by inducing an anti-viral response

Abstract

Abstract Background and Aim It is believed that the anti-tumor effect of DNA methylation inhibitors is mediated by re-activation of epigenetically silenced tumor suppressor genes in cancer cells. However, recent studies have proposed that the major anti-tumor effect of DNA methylation inhibitors is induction of interferon-related genes via dsRNAs-containing endogenous retroviruses. These studies used conventional cancer cell lines, which are maintained in 2D culture condition with serum-containing medium and are considered to be relatively artificial models of cancer cells. Recently, 3D culture system for stem cells known as organoid culture has been developed. Lgr5-positive stem cells form organoids that closely recapitulate the properties of original tissues. To investigate the effect of DNA demethylation on tumor organoids, we have established intestinal tumor organoids from tumors of ApcMin/+ (Min) mice and subjected them to 5-aza-2’-deoxycytidine (5-Aza-CdR) treatment and Dnmt1 knockdown. Methods Min mice were treated with 5-Aza-CdR (1 μg/body weight, n = 12) or PBS (n = 11) by subcutaneous injection weekly from 6 weeks of age. At 21 weeks of age, mice were dissected and number of intestinal polyps was counted. Stem cells were isolated from intestinal tumors of Min mice and maintained by organoid culture. Treatment with 5-Aza-CdR and lentivirus-mediated knockdown of Dnmt1 were performed in organoids derived from intestinal tumors. Expression profiles of genes after treatment with 5-Aza-CdR and Dnmt1-knockdown were analyzed. Results Treatment of Min mice with 5-Aza-CdR significantly reduced the average number of intestinal adenomas from 66 to 44 (male) and from 65 to 47 (female). The average number of large adenomas (≧3mm) in Min mice treated with 5-Aza-CdR was significantly decreased from 24 to11, whereas there was no significant difference in the average number of small adenomas (< 3mm). We successfully established organoids derived from intestinal tumors of Min mice by 3D culture with serum-free medium including epidermal growth factor (EGF) and Noggin. DNA demethylation induced by 5-Aza-CdR treatment and Dnmt1 knockdown significantly reduced the cell proliferation of the tumor organoids. Microarray analyses of the tumor organoids after 5-Aza-CdR treatment and Dnmt1 knockdown revealed that interferon-related genes including interferon regulatory factor 7 (Irf7) were activated by DNA demethylation. Gene ontology and pathway analyses clearly demonstrated that these genes activated by DNA demethylation are involved in the anti-viral response. Conclusions DNA demethylation suppresses the proliferation of intestinal tumor organoids by inducing an anti-viral response including activation of interferon-related genes. These findings suggest that treatment of colon cancers with DNA methylation inhibitors such as 5-Aza-CdR may be an effective therapeutic approach inhibiting cancer stem cells by immune response. Citation Format: Yoshimasa Saito, Kasumi Sakai, Toshihide Muramatsu, Toshiaki Nakaoka, Masaki Kimura, Hidetsugu Saito. Inhibition of DNA methylation suppresses intestinal tumor organoids by inducing an anti-viral response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2788.