Kuniaki Sato

Circulating exosomal microRNA-203 is associated with metastasis possibly via inducing tumor-associated macrophages in colorectal cancer

A primary tumor can create a premetastatic niche in distant organs to facilitate the development of metastasis. The mechanism by which tumor cells communicate with host cells to develop premetastatic niches is unclear. We focused on the role of microRNA (miR) signaling in promoting metastasis. Here, we identified miR-203 as a signaling molecule between tumors and monocytes in metastatic colorectal cancer (CRC) patients. Notably, high expression of serum exosomal miR-203, a major form in circulation, was associated with distant metastasis and an independent poor prognostic factor, whereas low expression in tumor tissues was a poor prognostic factor in CRC patients. We also found that exosomes carrying miR-203 from CRC cells were incorporated into monocytes and miR-203 could promote the expression of M2 markers in vitro, suggesting miR-203 promoted the differentiation of monocytes to M2-tumor-associated macrophages (TAMs). In a xenograft mouse model, miR-203-transfected CRC cells developed more liver metastasis compared to control cells. In conclusion, serum exosomal miR-203 expression is a novel biomarker for predicting metastasis, possibly via promoting the differentiation of monocytes to M2-TAMs in CRC. Furthermore, we propose the concept of site-dependent functions for miR-203 in tumor progression.

miR-146a Polymorphism (rs2910164) Predicts Colorectal Cancer Patients' Susceptibility to Liver Metastasis.

miR-146a plays important roles in cancer as it directly targets NUMB, an inhibitor of Notch signaling. miR-146a is reportedly regulated by a G>C polymorphism (SNP; rs2910164). This polymorphism affects various cancers, including colorectal cancer (CRC). However, the clinical significance of miR-146a polymorphism in CRC remains unclear. A total of 59 patients with CRC were divided into 2 groups: a CC/CG genotype (n = 32) and a GG genotype (n = 27), based on the miR-146a polymorphism. cDNA microarray analysis was performed using 59 clinical samples. Significantly enriched gene sets in each genotype were extracted using GSEA. We also investigated the association between miR-146a polymorphism and miR-146a, NUMB expression or migratory response in CRC cell lines. The CC/CG genotype was associated with significantly more synchronous liver metastasis (p = 0.007). A heat map of the two genotypes showed that the expression profiles were clearly stratified. GSEA indicated that Notch signaling and JAK/STAT3 signaling were significantly associated with the CC/CG genotype (p = 0.004 and p = 0.023, respectively). CRC cell lines with the pre-miR-146a/C revealed significantly higher miR-146a expression (p = 0.034) and higher NUMB expression and chemotactic activity. In CRC, miR-146a polymorphism is involved in liver metastasis. Identification of this polymorphism could be useful to identify patients with a high risk of liver metastasis in CRC.

Attenuated RND1 Expression Confers Malignant Phenotype and Predicts Poor Prognosis in Hepatocellular Carcinoma

ABSTRACTBackgroundThe RND1 gene encodes a protein that belongs to the Rho GTPase family, which regulates various cellular functions. Depletion of RND1 expression activates the oncogenic Ras signaling pathway. In this study, we aimed to clarify the clinical significance of RND1 expression in predicting prognosis and to investigate its biological role in human hepatocellular carcinoma (HCC).MethodsThe association between RND1 expression and clinical outcomes in patients with HCC was analyzed in three independent cohorts: 120 cases resected in our hospital; 370 cases in The Cancer Genome Atlas (TCGA); and 242 cases in GSE14520. Gene set enrichment analysis (GSEA) was also conducted. Finally, knockdown experiments were performed using small interfering RNA (siRNA) in vitro.ResultsIn all cohorts, RND1 expression was decreased as cancer progressed, and was affected by promoter methylation. In our HCC cases, the 5-year overall survival (OS) and recurrence-free survival of patients with low RND1 expression was significantly poorer than those of patients with high RND1 expression. TCGA and GSE14520 analyses provided similar results for OS. Multivariate analysis indicated that RND1 expression was an independent prognostic factor for OS in all three cohorts. Additionally, GSEA showed an inverse correlation between RND1 expression and the Ras signaling activity. In vitro, knockdown of RND1 expression resulted in significant increases in proliferation, invasion, and chemoresistance to cisplatin in HCC cells.ConclusionsReduced RND1 expression in HCC was associated with cancer progression, likely through regulation of the Ras signaling pathway, and may serve as a novel clinical biomarker for predicting prognosis in patients with HCC.

Identification of UHRF2 as a Negative Regulator of Epithelial-Mesenchymal Transition and Its Clinical Significance in Esophageal Squamous Cell Carcinoma

Objective: The involvement of epithelial-mesenchymal transition (EMT) in esophageal squamous cell carcinoma (ESCC) has not been fully elucidated. Here, we aimed to identify EMT-related genes associated with TGF-β in ESCC and to clarify the role of these genes in the progression of ESCC. Methods: EMT-related genes associated with TGF-β expression were identified in patients with ESCC using microarray analysis and public datasets. The effects of ubiquitin-like with PHD and ring finger domains 2 (UHRF2) expression were analyzed in ESCC cell lines. Cell proliferation and invasion were measured using MTT and invasion assays, respectively. UHRF2 mRNA expression was also analyzed in 75 ESCC specimens to determine the clinical significance of UHRF2 in ESCC. Results: Treatment of ESCC cell lines with TGF-β increased UHRF2 expression. UHRF2 overexpression increased CDH1 (E-cadherin) expression and decreased invasive capacity. The 75 ESCC specimens were divided into the UHRF2 high-expression group (n = 61) and the UHRF2 low-expression group (n = 14). Low UHRF2 expression was significantly correlated with vascular invasion (p = 0.034) and was an independent prognostic factor for poor prognosis (p = 0.005). Conclusion: UHRF2 may be a negative regulator of EMT and a novel prognostic biomarker for ESCC.

Abstract 1972: Identification of novel candidate driver genes of colorectal cancer on chromosome 7p

Background Colorectal cancer (CRC) is one of the most prevalent types of cancer. The high mortality rate of CRC is a serious problem. Hence it is urgently necessary to identify novel molecular target to improve the mortality rate. Amplification of chromosome 7p is frequent in CRC, and it has been considered to harbor driver genes that promote tumorigenesis or tumor progression by the gain of function. The aim of this study is to identify novel candidate driver genes on chromosome 7p and to clarify the clinical significance of their expression in CRC. Material and Methods 1. We selected the candidate genes that satisfied the following criteria using CRC data from The Cancer Genome Atlas (TCGA). 1) The DNA copy number and mRNA expression is positively correlated with each other, 2) overexpressed in the tumor tissues compared to the normal tissues. 2. The mRNA expression of the candidate genes was measured in 108 surgically-resected CRC tissues and the paired normal tissues in our hospital by quantitative RT-PCR. The differences of mRNA expression between CRC tissues and normal tissues were analyzed by Mann Whitney U-test. 3. Survival analysis between high and low expression group of the candidate genes was performed by Kaplan-Meier method. Correlation between the mRNA expression of the candidate genes and the clinicopathological factors were analyzed by Fisher’s exact test. 4. We performed Gene Set Enrichment Analysis (GSEA) in CRC data from TCGA to clarify the correlation between the candidate genes and gene sets that are associated with tumorigenesis or tumor progression. Results DEAD Box Helicase56 (DDX56), ATP-dependent RNA helicases involved in several aspects of RNA metabolism including mRNA splicing and transport, transcription, translation and remodeling of ribonucleoprotein complexes, was satisfied with the criteria. The expression of DDX56 was significantly higher in CRC tissues than in normal colon tissues (p Conclusions We identified DDX56 as a promising driver gene of CRC on chromosome 7p. DDX56 expression was positively associated with lymphatic invasion and distant metastasis, and was an independent poor prognostic factor. Furthermore, DDX56 may be involved in tumor progression through stimulating cell-cycle. DDX56 could be a therapeutic target as well as a poor prognostic biomarker in CRC. Note: This abstract was not presented at the meeting. Citation Format: Yuta Kouyama, Yushi Ogawa, Takaaki Masuda, Yukihiro Yoshikawa, Miwa Noda, Hiroaki Wakiyama, Kuniaki Sato, Sho Nambara, Qingjiang Hu, Shinya Kidogami, Tomoko Saito, Shotaro Sakimura, Naoki Hayashi, Yohsuke Kuroda, Shuhei Ito, Hidetoshi Eguchi, Koshi Mimori. Identification of novel candidate driver genes of colorectal cancer on chromosome 7p [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1972. doi:10.1158/1538-7445.AM2017-1972

Abstract 1843: Identification of peritoneal dissemination-related genes in gastric cancer

INTRODUCTION: Peritoneal dissemination (PD) is highly frequent and incurable metastasis in gastric cancer (GC). The mechanism causing PD is still poorly understood. In this study, we aimed to identify the genes responsible for PD using several large-scale public microarray databases. MATERIALS AND METHODS: First, we identified the candidate genes, which satisfied the all-4 outlines using microarray data as follow: 1) overexpressed in GC cell lines with high potential of PD; 2) overexpressed in GC patients with PD in Singapore from Duke-NUS Graduate Medical School; 3) overexpressed in tumor tissues in GC from TCGA database; 4) poor prognostic factor in GC from TCGA database. Next, we measured the expression of candidate genes by Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and performed clinicopathological studies of the expression in 146 Japanese GC patients who underwent surgery in our hospital. Finally, we performed gene set enrichment analysis (GSEA) with TCGA database to elucidate the correlation between candidate genes and gene sets that are associated with tumorigenesis or tumor progression in GC. Then, we examined the correlation between the candidate genes and one of the gene sets that were identified through GSEA by qRT-PCR in 5 GC cell lines (MKN7, KATO3, AGS, HSC39, 39As). RESULTS: 1. We identified Arl4c (ADP-ribosylation factor-like 4c) as a PD candidate gene by above our screening system. 2. The expression of Arl4c was significantly higher in tumor tissues than in corresponding normal tissues in Japanese GC patients (p 3. GSEA revealed that the expression of Arl4c positively correlated with epithelial-mesenchymal transformation (EMT) gene set. In the 5 GC cell lines, the expression of Arl4c negatively correlated with the expression of cdh1, which was well known as a down-regulated gene in EMT. CONCLUSION: Arl4c is a member of the ADP-ribosylation factor family of GTP-binding proteins and plays a role in cholesterol transport. Recent study revealed that Arl4c regulates YAP/TAZ pathway that is well known to induce EMT and promotes tumorigenesis in lung or colorectal cancer. Our results suggest that Arl4c should be involved in PD via EMT induced by the YAP/TAZ pathway, resulting in a poor prognostic marker in GC. Arl4c may be a novel biomarker and a therapeutic target for PD in GC. Citation Format: Qingjiang Hu, Shiya Kidogami, Sho Nambara, Hisateru Komatsu, Kuniaki Sato, Yushi Ogawa, Tomoko Saito, Shotaro Sakimura, Hidenari Hirata, Ryutaro Uchi, Naoki Hayashi, Tomohiro Iguchi, Takaaki Masda, Shuhei Ito, Hidetoshi Eguchi, Yoshihiko Maehara, Koshi Mimori. Identification of peritoneal dissemination-related genes in gastric cancer. [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 1843.