Naoto Iwai

Genome-wide DNA methylation analysis in hepatocellular carcinoma

Epigenetic changes as well as genetic changes are mechanisms of tumorigenesis. We aimed to identify novel genes that are silenced by DNA hypermethylation in hepatocellular carcinoma (HCC). We screened for genes with promoter DNA hypermethylation using a genome-wide methylation microarray analysis in primary HCC (the discovery set). The microarray analysis revealed that there were 2,670 CpG sites that significantly differed in regards to the methylation level between the tumor and non-tumor liver tissues; 875 were significantly hypermethylated and 1,795 were significantly hypomethylated in the HCC tumors compared to the non‑tumor tissues. Further analyses using methylation-specific PCR, combined with expression analysis, in the validation set of primary HCC showed that, in addition to three known tumor-suppressor genes (APC, CDKN2A, and GSTP1), eight genes (AKR1B1, GRASP, MAP9, NXPE3, RSPH9, SPINT2, STEAP4, and ZNF154) were significantly hypermethylated and downregulated in the HCC tumors compared to the non-tumor liver tissues. Our results suggest that epigenetic silencing of these genes may be associated with HCC.

EVI1, a target gene for amplification at 3q26, antagonizes transforming growth factor‐β‐mediated growth inhibition in hepatocellular carcinoma

EVI1 (ecotropic viral integration site 1) is one of the most aggressive oncogenes associated with myeloid leukemia. We investigated DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines using a high‐density oligonucleotide microarray. We found that a novel amplification at the chromosomal region 3q26 occurs in the HCC cell line JHH‐1, and that MECOM (MDS1 and EVI1 complex locus), which lies within the 3q26 region, was amplified. Quantitative PCR analysis of the three transcripts transcribed from MECOM indicated that only EVI1, but not the fusion transcript MDS1–EVI1 or MDS1, was overexpressed in JHH‐1 cells and was significantly upregulated in 22 (61%) of 36 primary HCC tumors when compared with their non‐tumorous counterparts. A copy number gain of EVI1 was observed in 24 (36%) of 66 primary HCC tumors. High EVI1 expression was significantly associated with larger tumor size and higher level of des‐γ‐carboxy prothrombin, a tumor marker for HCC. Knockdown of EVI1 resulted in increased induction of the cyclin‐dependent kinase inhibitor p15INK4B by transforming growth factor (TGF)‐β and decreased expression of c‐Myc, cyclin D1, and phosphorylated Rb in TGF‐β‐treated cells. Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability. Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF‐β‐mediated growth inhibition of HCC cells.

Loss of PAR-3 protein expression is associated with invasion, lymph node metastasis, and poor survival in esophageal squamous cell carcinoma

Disrupted cell polarity is a feature of epithelial cancers. The partitioning defective 3 (PAR-3) protein, a key component of the PAR complex that regulates the polarization of cells, is involved in tight junction formation at epithelial cell-cell contacts. Our previous study detected a homozygous deletion of the PAR-3 gene in esophageal squamous cell carcinoma (ESCC) cell lines and frequent copy number loss of the PAR-3 gene in primary ESCC. Here, we aimed to investigate the clinicopathological relevance of altered expression of the PAR-3 protein in primary ESCC. We immunohistochemically analyzed expression of the PAR-3 protein, as well as that of other tight junction proteins, ZO-1 and claudin-1, in 74 primary ESCCs. While the PAR-3 protein was expressed in the cytoplasm of basal cells, it was localized on the plasma membrane of suprabasal cells of normal squamous epithelium of the esophagus. Of the 74 ESCC tumors, 20 (27%), 11 (15%), and 13 (18%) were negative for PAR-3, ZO-1, and claudin-1 proteins, respectively. Negative PAR-3 protein expression, but not negative ZO-1 or claudin-1 expression, was significantly associated with deeper tumor invasion (P<.01), positive lymph node metastasis (P=.03), and advanced tumor stage (P=.01). Patients with PAR-3-negative tumors showed marginally significantly shorter overall survival after surgery than those with PAR-3-positive tumors (P=.053). In conclusion, these results suggest that PAR-3 protein expression is frequently lost in primary ESCC and that loss of the PAR-3 protein is associated with aggressive clinicopathological features of ESCC.

Stenotic Ischemic Enteritis with Concomitant Hepatic Portal Venous Gas and Pneumatosis Cystoides Intestinalis: A Case Report

A 69-year-old man was admitted to a hospital with complaints of abdominal pain. Computed tomography showed hepatic portal venous gas and pneumatosis cystoides intestinalis. Conservative treatment was effective; however, after discharge, he developed complaints of vomiting. Fluoroscopic enteroclysis revealed a stricture in the jejunum necessitating admission to our hospital. Transoral balloon-assisted enteroscopy showed a circumferential ulcer with a stricture. The stricture was surgically resected, and a histopathological examination was consistent with ischemic enteritis. Stenotic ischemic enteritis should be considered among the differential diagnoses in a patient presenting with hepatic portal venous gas and pneumatosis cystoides intestinalis showing small intestinal obstruction.

Impact of the Charlson comorbidity index and prognostic nutritional index on prognosis in patients with early gastric cancer after endoscopic submucosal dissection

With the aging of society, comorbidities or nutritional status are assessed prior to endoscopic submucosal dissection (ESD) for early gastric cancer (EGC). However, it is uncertain which factors are important for predicting prognosis in EGC patients after ESD. Thus, we aimed to evaluate clinical outcomes of ESD for EGC, with respect to comorbidities or nutritional status.

Abstract 2539: MiR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers with high mortality worldwide. MicroRNAs (miRNAs) are small non-coding RNAs that have been used as cancer-related biomarkers and expected to be therapeutic agents. We performed genome-wide miRNA expression profiling of paired HCC tumors and non-tumorous liver tissues from patients with primary HCCs using the miRNA microarray (Agilent). We found that miR-96-5p was most significantly up-regulated in HCC tumors compared to non-tumor tissues. Although miR-96-5p is suggested to be an oncogenic miRNA, the function of miR-96-5p remains largely unknown. We identified the caspase-9 gene (CASP9) as a novel target of miR-96-5p, in addition to the forkhead box O1 gene (FOXO1) which is the known target of it. Caspase-9 protein is thought to play a central role in apoptosis and to be a tumor suppressor. Overexpression of miR-96-5p decreased caspase-9 protein expression and resulted in resistance to apoptosis induced by doxorubicin and UV in HCC cells. Our results suggested that miR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis through decreasing caspase-9 expression in HCC. Citation Format: Naoto Iwai, Kohichiroh Yasui, Akira Tomie, Kei Teasaki, Tomoko Kitaichi, Osamu Dohi, Yasuyuki Gen, Yoshito Ito. MiR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis in hepatocellular carcinoma [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 2539. doi:10.1158/1538-7445.AM2017-2539

Abstract 1099: Up-regulated miR-96-5p inhibits apoptosis by targeting FOXO1 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of cancer death worldwide, especially in Asia and sub-Saharan Africa. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and can act as tumor suppressors or oncogenes. To identify miRNA involved in HCC, we performed a genome-wide miRNA gene expression analysis using the human miRNA microarray (Agilent) in paired tumor and non-tumor tissues from patients with primary HCC. The array-based miRNA expression profiles were validated by quantitative PCR. We identified miR-96-5p as the most significantly upregulated miRNA in HCC tumors. Although miR-96-5p has been recognized as an oncogenic miRNA, its role in the pathogenesis of HCC remains unknown. Our study showed that the inhibition of miR-96-5p reduced proliferation of HCC cells and induced apoptosis. Furthermore, we found that the forkhead box O1 (FOXO1) gene was a target of miR-96-5p. FOXO1, a transcription factor, plays an essential role in cell fate decisions. FOXO1 can induce apoptosis through mitochondria-dependent and mitochondria-independent pathways. Collectively, our results suggested that miR-96-5p may function as an oncogenic miRNA through inhibiting apoptosis by targeting FOXO1 in HCC. Citation Format: Naoto Iwai, Kohichiroh Yasui, Akira Tomie, Tomoko Kitaichi, Osamu Dohi, Yasuyuki Gen, Yoshito Itoh. Up-regulated miR-96-5p inhibits apoptosis by targeting FOXO1 in hepatocellular carcinoma. [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 1099.