Shinya Kidogami

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.

Clinical significance of ZNF750 gene expression, a novel tumor suppressor gene, in esophageal squamous cell carcinoma

The present authors previously identified a novel candidate tumor suppressor gene, zinc finger protein 750 (ZNF750), in esophageal squamous cell carcinoma (ESCC) (1). The present study aimed to clarify the clinical significance of ZNF750 expression in ESCC. The association between ZNF750 DNA mutation status and the mRNA expression was examined by whole exome sequence analysis and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The expression of ZNF750 in 76 patients with ESCC (Kyushu University Beppu Hospital) was measured using immunohistochemistry and RT-qPCR. Using this dataset, the association between ZNF750 mRNA expression and clinicopathological factors was examined. Additionally, survival analysis was performed using datasets from the Kyushu University Beppu Hospital and The Cancer Genome Atlas (TCGA). The biological effects of ZNF750 expression were explored using gene set enrichment analysis (GSEA) and were validated using datasets from the Cancer Cell Line Encyclopedia (CCLE) and the Kyushu University Beppu Hospital. ZNF750 expression analyses demonstrated that ZNF750 mRNA expression was lower in patients with the DNA mutations compared with those without the mutations (P<0.05), and ZNF750 expression was downregulated in tumor tissues compared with normal tissues (P<0.00005). In the clinicopathological analysis, the low ZNF750 expression group exhibited a higher incidence of undifferentiated histology (P<0.05) compared with the high expression group. The low ZNF750 expression group exhibited a poorer prognosis in the Kyushu and TCGA datasets (P<0.0005 and P<0.05, respectively). GSEA indicated that ZNF750 expression was significantly correlated with epithelial differentiation in ESCC. This was confirmed using the datasets from CCLE and the Kyushu University Beppu Hospital by analyzing the levels of small proline rich protein 1A mRNA, an epithelial differentiation-associated gene. In conclusion, the results of the present study suggested that ZNF750 serves a role as a tumor suppressor; potentially via regulating epithelial differentiation and that it may be a promising biomarker of poor outcomes in ESCC.

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 149: Integrated analysis of intratumor heterogeneity with genetic and epigenetic aberrations during colorectal cancer evolution

[background] Intratumor heterogeneity (ITH) is presumably generated by branching clonal evolution of cancer cells. Recently, a multiregional sequencing approach, which sequences DNA sampled from geographically separated regions of a single tumor, has revealed branched evolution and ITH. In this study, we present genetic and epigenetic analysis of ITH in a series of nine colorectal cancers [material and method] We conducted analysis of samples from geographically separated regions from nine colorectal tumors. From each of the nine tumor, we obtained 5-21 multiregional samples, which were 75 samples in total, together with 9 paired normal mucosa samples. For two cases, samples from liver metastases were obtained. We performed exome sequencing and copy number (CN), methylation, and mRNA expression array profiling. [Result] Each of the multiregional mutation profiles harbored founder and progressor mutations; founder mutations are shared by all regions while progressor mutations are not. We found that mutations in well-known driver genes such as APC, KRAS, and FBWX7 were acquired as founder mutations, while mutations in PIK3CA recurrently occurred as progressor mutations, suggesting that PIK3CA mutations are a late event in the evolution of colorectal cancer. We counted each category of mutation and then identified a correlation between the number of founder mutations and the age of the patients. Our analysis showed that C > T transitions at CpG sites are more prominent in founder mutations than in progressor mutations. Then, the multiregional CN profiles showed that amplifications of 7p, 13q, 10q, 20p, and 20q frequently occurred across all samples in multiple tumors, namely as founder CN alterations. Finally, our data showed that hypermethylations in CpG islands were more prominent in founder methylations than in progressor methylations, suggesting that CpG island hypermethylations mainly occur in the early phase of colorectal cancer evolution. [Conclusion] In this study, our integrated analysis revealed not only extensive ITH, but also the evolutionary histories of the nine tumors. In particular, by focusing on founder and progressor mutations, we identified clues for decoding the life history of the tumors. Citation Format: Shinya Kidogami, Atsushi Niida, Ryutaro Uchi, Yusuke Takahashi, Masaki Mori, Satoru Miyano, Koshi Mimori. Integrated analysis of intratumor heterogeneity with genetic and epigenetic aberrations during colorectal cancer evolution. [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 149.