Inhibition of DNA Methylation Suppresses Intestinal Tumor Organoids by Inducing an Anti-Viral Response.

Yoshimasa Saito,Toshiro Sato,Toshiaki Nakaoka,Hidetsugu Saito,Masaki Kimura,Takanori Kanai,Kohta Toshimitsu,Toshihide Muramatsu,Kasumi Sakai

doi:10.1038/srep25311

Abstract

Recent studies have proposed that the major anti-tumor effect of DNA methylation inhibitors is induction of interferon-responsive genes via dsRNAs-containing endogenous retroviruses. Recently, a 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 organoids from intestinal tumors of ApcMin/+ (Min) mice and subjected them to 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment and Dnmt1 knockdown. 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-responsive genes 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. These findings indicate that DNA demethylation suppresses the proliferation of intestinal tumor organoids by inducing an anti-viral response including activation of interferon-responsive genes. Treatment with DNA methylation inhibitors to activate a growth-inhibiting immune response may be an effective therapeutic approach for colon cancers.

Highlights

Tumor stem cells with self-renewal and multipotential capacity play critical roles in malignant tumors that show a marked capacity for metastasis and invasion
Several studies have demonstrated that DNA methylation inhibitors such as 5-Aza-CdR and zebularine suppress intestinal tumors in Min mice[17,18]
The tumor organoids derived from Apc-deficient tumors can be stably maintained in culture medium without Rspo[1] and form cystic organoid structures, because Apc loss constitutively activates the Wnt signaling pathway in tumor cells

Summary

Introduction

Tumor stem cells with self-renewal and multipotential capacity play critical roles in malignant tumors that show a marked capacity for metastasis and invasion. Lgr[5], a member of the Wnt signaling pathway, has been identified as a new molecular marker of stem cells in the small intestine, colon, stomach, liver and pancreas[5,6,7,8,9,10]. A newly developed 3D culture system allows Lgr5-positive stem cells to form budding cyst-like structures (organoids) that resemble the properties of original tissues[5]. This type of 3D culture uses serum-free medium that includes only defined factors such as R-spondin 1 (Rspo1), epidermal growth factor (EGF) and noggin. To investigate the effect of DNA demethylation on tumor organoids, we established intestinal tumor organoids derived from tumors of ApcMin/+ (Min) mice and subjected them to treatment with 5-Aza-CdR and knockdown of Dnmt[1]

Methods

Results

Conclusion