Toshihide Muramatsu

Silencing of microRNA‐122 is an early event during hepatocarcinogenesis from non‐alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) has emerged as a common cause of chronic liver disease and virus‐independent hepatocellular carcinoma (HCC) in patients with obesity, diabetes, and metabolic syndrome. To reveal the molecular mechanism underlying hepatocarcinogenesis from NASH, microRNA (miRNA) expression profiles were analyzed in STAM mice, a NASH‐HCC animal model. MicroRNA expression was also examined in 42 clinical samples of HCC tissue. Histopathological images of the liver of STAM mice at the ages of 6, 8, 12, and 18 weeks showed findings compatible with fatty liver, NASH, liver cirrhosis (LC), and HCC, respectively. Expression of miR‐122 in non‐tumor LC at the age of 18 weeks was significantly lower than that in LC at the age of 12 weeks. Expression of miR‐122 was further decreased in HCCs relative to non‐tumor LC at the age of 18 weeks. Expression of miR‐122 was also decreased in clinical samples of liver tissue showing macrovesicular steatosis and HCC, being consistent with the findings in the NASH model mice. DNA methylation analysis revealed that silencing of miR‐122 was not mediated by DNA hypermethylation of the promoter region. These results suggest that silencing of miR‐122 is an early event during hepatocarcinogenesis from NASH, and that miR‐122 could be a novel molecular marker for evaluating the risk of HCC in patients with NASH.

Inhibitors of enhancer of zeste homolog 2 (EZH2) activate tumor-suppressor microRNAs in human cancer cells

Enhancer of zeste homolog 2 (EZH2) enhances tumorigenesis and is commonly overexpressed in several types of cancer. To investigate the anticancer effects of EZH2 inhibitors, microRNA (miRNA) expression profiles were examined in gastric and liver cancer cells treated with suberoylanilide hydroxamic acid (SAHA) and 3-deazaneplanocin A (DZNep). We confirmed that SAHA and DZNep suppressed EZH2 expression in AGS and HepG2 cells and inhibited their proliferation. The results of microarray analyses demonstrated that miR-1246 was commonly upregulated in cancer cells by treatment with SAHA and DZNep. MiR-302a and miR-4448 were markedly upregulated by treatment with SAHA and DZNep, respectively. DYRK1A, CDK2, BMI-1 and Girdin, which are targets of miR-1246, miR-302a and miR-4448, were suppressed by treatment with SAHA and DZNep, leading to apoptosis, cell cycle arrest and reduced migration of AGS and HepG2 cells. ChIP assay revealed that SAHA and DZNep inhibited the binding of EZH2 to the promoter regions of miR-1246, miR-302a and miR-4448. These findings suggest that EZH2 inhibitors such as SAHA and DZNep exert multiple anticancer effects through activation of tumor-suppressor miRNAs.

Suppressive effect of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) on hepatitis C virus replication

The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) has a clinical promise for treatment of cancer including hepatocellular carcinoma (HCC). To investigate effect of SAHA on hepatitis C virus (HCV) replication, we treated the HCV replicon cell OR6 with SAHA. HCV replication was significantly inhibited by SAHA at concentrations below 1 μM with no cellular toxicity. Another HDAC inhibitor, tricostatin A, also showed reduction of HCV replication. The microarray analysis and quantitative RT‐PCR demonstrated up‐regulation of osteopontin (OPN) and down‐regulation of apolipoprotein‐A1 (Apo‐A1) after SAHA treatment. Direct gene induction of OPN and knockdown of Apo‐A1 also showed reduction of HCV replication. The liver specific microRNA‐122, which is involved in HCV replication, was not affected by SAHA treatment. These results suggest that SAHA has suppressive effect on HCV replication through alterations of gene expression such as OPN and Apo‐A1 in host cells. Epigenetic treatment with HDAC inhibitors may be a novel therapeutic approach for diseases associated with HCV infection such as chronic hepatitis, liver cirrhosis, and HCC. J. Cell. Biochem. 114: 1987–1996, 2013.

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

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.

Induction of differentiation of intrahepatic cholangiocarcinoma cells to functional hepatocytes using an organoid culture system

Intrahepatic cholangiocarcinoma (IHCC) is a highly aggressive malignancy with a poor prognosis. It is thought to originate from cholangiocytes, which are the component cells of intrahepatic bile ducts. However, as patients with viral hepatitis often develop IHCC, it has been suggested that transformed hepatocytes may play a role in IHCC development. To investigate whether IHCC cells can be converted to functional hepatocytes, we established organoids derived from human IHCC and cultured them under conditions suitable for hepatocyte differentiation. IHCC organoids after hepatocyte differentiation acquired functions of mature hepatocytes such as albumin secretion, bile acid production and increased CYP3A4 activity. Studies using a mouse model of IHCC indicate that Wnt3a derived from macrophages recruited upon inflammation in the liver may promote the malignant transformation of hepatocytes to IHCC cells. The results of the present study support the recently proposed hypothesis that IHCC cells are derived from hepatocytes.

Abstract 4089: Establishment and long-term in vitro culture of organoids derived from human biliary tract carcinomas

Background Biliary tract carcinomas (BTCs) are epithelial malignancies arising in the region between the intrahepatic bile ducts and the ampulla of Vater at the distal end of the common bile duct. Although patients with inoperable BTCs generally receive a chemotherapy regimen of gemcitabine and cisplatin, the effect of these drugs is limited and the 5-year survival rates of patients are very low. The newly developed 3D culture system known as “organoid culture” allows long-term expansion of stem cells into budding cyst-like structures (organoids) with properties resembling those of the original tissues. The aim of this study is to establish in vitro preclinical models for patients with BTCs using the organoid culture technology. Methods We established organoids using cancer tissues obtained from patients with BTCs. Alterations of genome, gene expression profiles including microRNAs and drug sensitivity were analyzed in BTC organoids. Results We were successful in establishment and long-term in vitro culture of five organoid lines derived from human BTCs including intrahepatic cholangiocarcinoma, gallbladder cancer and neuroendocrine carcinoma of the ampulla of Vater. These BTC organoids can be stably cultured over a year, whereas organoids derived from non-cancer bile duct and gallbladder tissues ceased proliferating after 15 passages or six months. HE 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4089.

Cluster microRNAs miR-194 and miR-215 suppress the tumorigenicity of intestinal tumor organoids

Tumor stem cells with self‐renewal and multipotent capacity play critical roles in the initiation and progression of cancer. Recently, a new 3‐D culture system known as organoid culture has been developed, allowing Lgr5‐positive stem cells to form organoids that resemble the properties of original tissues. Here we established organoids derived from intestinal tumors of Apcmin/+ mice and normal intestinal epithelia of C57BL/6J mice and investigated the roles of microRNA (miRNA) in intestinal tumor organoids. The results of microarray analyses revealed that expression of the cluster miRNAs, miR‐194 and miR‐215 was markedly suppressed in intestinal tumor organoids in comparison with organoids derived from normal intestinal epithelia. Enforced expression of miR‐194 resulted in inhibition of E2f3, a positive regulator of the cell cycle and growth suppression of intestinal tumor organoids. In addition, enforced expression of miR‐215 suppressed the cancer stem cell signature through downregulation of intestinal stem cell markers including Lgr5. These findings indicate that the miRNA cluster including miR‐194 and miR‐215 plays important roles in suppressing the growth and attenuating the stemness of intestinal tumor organoids.

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

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 ( 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.

Abstract 3112: Decrease of microRNA-122 is a key event during hepatocarcinogenesis from non-alcoholic steatohepatitis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background and Aim: Despite improvement in the treatment of viral infection, the incidence of hepatocellular carcinoma (HCC) worldwide is still high. Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver diseases and is known to develop liver cirrhosis (LC) and HCC in patients with obesity, diabetes, and metabolic syndrome. NASH may account for a large proportion of virus-independent HCC in developed countries. The aim of this study was to reveal the molecular mechanism underlying hepatocarcinogenesis from NASH, especially in the change of microRNA (miRNA) expression during carcinogenesis in an animal model. Methods and Results: MiRNA expression profiles were analyzed in STAM® mice, a NASH-HCC animal model. MicroRNA expression was also examined in 42 clinical samples from patients with HCC. Histopathology of the liver in STAM® mice at the ages of 6, 8, 12, and 18-weeks showed findings compatible with simple fatty liver, NASH, LC, and HCC, respectively. The result of miRNA expression profile showed that the liver-specific miRNA, miR-122, significantly decreased in non-tumor LC at the age of 18-weeks than that in LC at the age of 12-weeks. Expression of miR-122 was further decreased in HCCs than non-tumor LC at the age of 18-weeks. Expression of miR-122 was also decreased in clinical samples of liver tissue showing macrovesicular steatosis and HCC, being consistent with the findings in the NASH model mice. To further investigate the mechanism of decrease in miR-122 expression, the promoter region of miR-122 in human cell lines, HepG2 and HuH7, clinical samples and mice livers was analyzed. In vitro study using human cell lines revealed that the miR-122 promoter region was CpG-poor but DNA methylation of the specific region in miR-122 promoter, where peroxisome proliferator activated receptor-γ is associated, was critical for regulation of miR-122 expression, which was demonstrated by treatment with a DNA methylation inhibitor. However, the average levels of DNA methylation in the miR-122 promoter region were significantly reduced in HCC tissues than non-tumor liver tissues in human clinical samples. And also there was no significant difference in the level of DNA methylation between non-tumor LC and HCC in mice samples. These results suggested that that silencing of miR-122 was not mediated by DNA hypermethylation of the promoter region in this NASH-HCC model. Conclusion: Thus, although epigenetic regulation of miR-122 expression was still unclear, the results in the present study indicated that silencing of miR-122 was an early event during hepatocarcinogenesis from NASH, and that miR-122 could be a novel molecular marker for evaluating the risk of HCC in patients with NASH. Citation Format: Hidetsugu Saito, Yoko Takaki, Azusa Takasugi, Shoji Yamada, Toshihide Muramatsu, Masaki Kimura, Kazuo Sugiyama, Hiromu Suzuki, Yae Kanai, Yoshimasa Saito. Decrease of microRNA-122 is a key event during hepatocarcinogenesis from non-alcoholic steatohepatitis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3112. doi:10.1158/1538-7445.AM2015-3112

Tu1900 MiR-1246 and Mir-302 Are Novel Targets of Epigenetic Therapy With Dznep and SAHA in Human Cancer Cells

Introduction Epigenetic therapy with DNA methylation inhibitors and histone deacetylase (HDAC) inhibitors holds clinical promise for the treatment of human malignancies. MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of various target genes and play critical roles in cancer. We have proposed that epigenetic activation of tumor suppressor miRNAs can be a novel therapeutic approach for human cancers (Cancer Cell 9: 435, 2006). Recently, 3-deazaneplanocin A (DZNep) was discovered to inhibit a polycomb group protein, enhancer of zeste homologue 2 (EZH2), which has activity for trimethylation of histone H3K27. To investigate the molecular mechanisms underlying the effect of epigenetic therapy with DZNep and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) against human cancer, miRNA expression profiles were analyzed. Materials and Methods AGS human gastric cancer cells and HepG2 human liver cancer cells were treated with DZNep and SAHA. MiRNA expression profiles were analyzed by microarray analysis and TaqMan quantitative RT-PCR. Cell proliferation was analyzed by cell counting, and target genes of miRNAs were analyzed by Western blotting. Levels of apoptosis and the cell cycle were analyzed by flow cytometry. Results The results of microarray and quantitative RT-PCR analyses revealed that miR-1246 was up-regulated by treatment with DZNep in both AGS and HepG2 cells, and that miR-302 was up-regulated by treatment with SAHA in AGS cells. Treatment with DZNep or SAHA reduced the cell proliferation activity of both AGS and HepG2 cells, and this reduction was more marked when both drugs were used in combination. The anti-apoptotic factor DYRK1A, which was recently identified as one of the targets of miR-1246, was significantly suppressed by treatment with DZNep and SAHA, resulting in apoptosis of AGS andHepG2 cells. In addition, treatment of AGS cells with DZNep and SAHA suppressed CDK2 and BMI-1, which were recently identified as the targets of miR-302, inducing cell cycle (G1/S) arrest of AGS cells. Conclusions These findings suggest that DZNep is a promising drug for the treatment of gastric and liver cancers via activation of miR-1246. DZNep and SAHA may synergistically suppress the proliferation of gastric cancer cells via activation of miR-1246 and miR-302. MiR-1246 and miR-302 are novel targets of epigenetic therapy with DZNep and SAHA in human cancer cells.