Shuji Nomoto

Reduced Expression of Adherens Junctions Associated Protein 1 Predicts Recurrence of Hepatocellular Carcinoma After Curative Hepatectomy

BackgroundHepatocellular carcinoma (HCC) frequently recurs after curative resection. Therefore, the availability of sensitive biomarkers for progression and recurrence is essential for managing patients’ clinical course. Adherens junctions associated protein 1 (AJAP1) may serve this purpose, because it mediates activities of tumor cells.MethodsAJAP1 mRNA levels and those of genes encoding potential interacting proteins, such as SRC in HCC cell lines, and 144 pairs of resected liver tissues were determined as well as the methylation status of the AJAP1 promoter and copy number changes at AJAP1 locus. The expression pattern of AJAP1 protein was evaluated using immunohistochemistry.ResultsAJAP1 mRNA levels varied among nine HCC cell lines, and AJAP1 expression was reactivated after demethylation of its promoter. AJAP1 mRNA levels correlated inversely with those of SRC in HCC cell lines and tissues. AJAP1 mRNA levels were suppressed in HCC tissues. The expression pattern of AJAP1 correlated significantly with that of AJAP1 mRNA. Low levels of AJAP1 mRNA in patients with HCC associated significantly with elevated levels of tumor markers, larger tumor size, serosal infiltration, vascular invasion, hypermethylation of the AJAP1 promoter, and copy number loss at AJAP1 locus. Patients with low levels of AJAP1 expression were more likely to experience shorter disease-free survival (DFS), and multivariate analysis identified low AJAP1 expression as an independent factor for predicting DFS.ConclusionsAJAP1 may function as a key regulatory molecule associated with the recurrence of HCC. Hypermethylation of the AJAP1 promoter is a key regulatory mechanism controlling AJAP1 expression.

Translational implication of Kallmann syndrome-1 gene expression in hepatocellular carcinoma.

Accumulation of epigenetic alterations causes inactivation of tumor suppressors and contributes to the initiation and progression of hepatocellular carcinoma (HCC). Identification of methylated genes is necessary to improve our understanding of the pathogenesis of HCC and develop novel biomarkers and therapeutic targets. The Kallmann syndrome-1 (KAL1) gene encodes an extracellular matrix-related protein with diverse oncological functions. However, the function of KAL1 in HCC has not been examined. We investigated the methylation status of the KAL1 promoter region in HCC cell lines, and evaluated KAL1 mRNA levels and those of genes encoding potential interacting cell adhesion factors. KAL1 mRNA expression level was heterogeneous in nine HCC cell lines, and reactivation of KAL1 mRNA expression was observed in cells with promoter hypermethylation of KAL1 gene after demethylation. In addition, KAL1 mRNA levels inversely correlated with those of ezrin in all nine HCC cell lines. KAL1 expression levels in 144 pairs of surgically-resected tissues were determined and correlated to clinicopathological parameters. KAL1 mRNA level was independent of the background liver status, whereas HCC tissues showed significantly lower KAL1 mRNA levels than corresponding noncancerous liver tissues. Downregulation of KAL1 mRNA in HCC was significantly associated with malignant phenotype characteristics, including elevated tumor markers, larger tumor size, vascular invasion, and hypermethylation of KAL1. Patients with downregulation of KAL1 were more likely to have a shorter overall survival than other patients, and multivariate analysis identified downregulation of KAL1 as an independent prognostic factor (hazard ratio 2.04, 95% confidence interval 1.11-3.90, P=0.022). Our results indicated that KAL1 may act as a putative tumor suppressor in HCC and is inactivated by promoter hypermethylation. KAL1 may serve as a biomarker of malignant phenotype of HCC.

CCNJ detected by triple combination array analysis as a tumor-related gene of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has a high likelihood of recurrence and a poor prognosis. To detect cancer-related genes of HCC, we developed a new technique: triple combination array analysis, consisting of a methylation array, a gene expression array and a single nucleotide polymorphism array. A surgical specimen obtained from a 68-year-old female HCC patient was analyzed using triple combination array, which identified cyclin J (CCNJ) as a candidate cancer-related gene of HCC. Subsequently, samples from 85 HCC patients were evaluated for CCNJ promoter hypermethylation and expression status using methylation-specific PCR (MSP) and quantitative reverse transcriptase RT-PCR, respectively. CCNJ was found to be hypermethylated (methylation value, 0.906; range, 0-1.0) in cancer tissue, compared with adjacent non-cancerous tissue (0.112) using a methylation array. MSP revealed that CCNJ was hypermethylated in 67 (78.8%) of the tumor samples. CCNJ expression was significantly decreased in cases with hypermethylation (P<0.0001). Furthermore, cases with both promoter hypermethylation and decreased expression of CCNJ in the tumor tissue had a worse overall survival than the other cases (P=0.0383). In conclusion, our results indicated that CCNJ could be a novel prognostic marker of HCC, and this study indicated that triple combination array analysis was effective in detecting new tumor-related genes and their mechanisms.

Genomic-Wide Analysis with Microarrays in Human Oncology

DNA microarray technologies have advanced rapidly and had a profound impact on examining gene expression on a genomic scale in research. This review discusses the history and development of microarray and DNA chip devices, and specific microarrays are described along with their methods and applications. In particular, microarrays have detected many novel cancer-related genes by comparing cancer tissues and non-cancerous tissues in oncological research. Recently, new methods have been in development, such as the double-combination array and triple-combination array, which allow more effective analysis of gene expression and epigenetic changes. Analysis of gene expression alterations in precancerous regions compared with normal regions and array analysis in drug-resistance cancer tissues are also successfully performed. Compared with next-generation sequencing, a similar method of genome analysis, several important differences distinguish these techniques and their applications. Development of novel microarray technologies is expected to contribute to further cancer research.

Suppression of SAMSN1 Expression is Associated with the Malignant Phenotype of Hepatocellular Carcinoma.

BackgroundIdentification of molecular markers for sensitive detection of hepatocellular carcinoma (HCC) is required to achieve efficacious personalized therapy.MethodsWe focused here on SAM domain, SH3 domain, and nuclear localization signals 1 (SAMSN1) and investigated expression and methylation status of SAMSN1 in HCC cell lines and 144 pairs of surgical specimens.ResultsSAMSN1 was expressed at significantly lower levels in tumor tissue compared with the corresponding noncancerous tissues of patients with HCC. Analysis of HCC cell lines revealed that hypermethylation of the SAMSN1 promoter correlated with decreased expression of SAMSN1 mRNA. Furthermore, treating cells with a DNA-demethylating drug increased SAMSN1 transcription. The levels of SAMSN1 mRNA in noncancerous liver were not affected by background liver inflammation or fibrosis. Moreover, the levels of SAMSN1 mRNA in HCC tissues inversely correlated with tumor size and preoperative levels of proteins induced by vitamin K absence. The clinical significance of SAMSN1 was further indicated by the correlation between its decreased expression in patients with HCC and their shorter overall and recurrence-free survival as well as recurrence following initial resection. Moreover, multivariate analysis identified SAMSN1 as an independent prognostic factor of HCC progression. The expression pattern of SAMSN1 correlated significantly with that of SAMSN1 mRNA, making it possible to use PCR techniques to readily quantitate SAMSN1 expression in tumors.ConclusionsOur findings indicate that inhibition of SAMSN1 transcription through DNA hypermethylation may influence the progression of HCC and thus represent a novel biomarker of the phenotype of HCC cells.

Search for useful biomarkers in hepatocellular carcinoma, tumor factors and background liver factors (Review)

Hepatocarcinogenesis is a complex and multistep process that involves the accumulation of genetic and epigenetic alterations in regulatory genes. To understand the development of hepatocellular carcinoma (HCC), current research has utilized improved array technologies. The identification of cancer-related molecules could lead to the development of novel molecular targets for treatment and biomarkers for predicting prognosis. However, prognostic prediction is insufficient when considering only tumor factors, since hepatocarcinogenesis is also greatly influenced by the status of the background liver. Clinical background liver factors, such as the presence of chronic active hepatitis or cirrhosis, are well known as risk factors for developing HCC. In contrast, genetic or epigenetic background liver factors remain unknown, albeit those are important to understand the developing process of HCC. Investigating background liver factors could contribute to the development of carcinogenic markers of HCC and to the prevention of the development of HCC. In the present study, we review the currently identified tumor factors and background liver factors from a molecular biological viewpoint and also introduce our combination array analysis.

Prognostic relevance of SAMSN1 expression in gastric cancer

The prognosis for patients with advanced gastric cancer (GC) remains poor. The identification of biomarkers relevant to the recurrence and metastasis of GC is advantageous for stratifying patients and proposing novel molecular targets. In the present study the oncological roles of SAM domain, SH3 domain and nuclear localization signals 1 (SAMSN1), a mediator of B-cell function, were elucidated in GC. The expression and methylation status of SAMSN1 were investigated in a panel of 11 GC cell lines. Immunohistochemical staining was performed to determine the pattern of SAMSN1 protein expression in gastric tissues. The prognostic impact of SAMSN1 expression was determined by analyzing 175 pairs of surgically resected gastric tissues. A marked decrease in the level of SAMSN1 mRNA was detected in 8/11 GC cell lines as compared with that in a non-transformed intestinal epithelium cell line (FHs 74) without promoter methylation. The mean expression level of SAMSN1 mRNA was reduced in GC tissues compared with normal adjacent tissues, an observation that was independent of tumor differentiation. The pattern of SAMSN1 protein expression was significantly correlated with that of SAMSN1 mRNA. Low SAMSN1 mRNA expression was significantly associated with tumor size (>60 mm; P=0.026) and shorter overall survival times (P=0.004). Multivariate analysis identified low SAMSN1 mRNA expression as an independent prognostic factor for poor overall survival (hazard ratio, 1.80; 95% confidence interval, 1.07-3.05; P=0.025). The difference in survival between the low and high SAMSN1 expression groups was more marked in patients with stage II/III GC compared to those with stage IV GC. In patients with stage II/III GC who underwent curative surgery, low SAMSN1 expression was associated with reduced disease free survival times. The results of the present study indicate that downregulation of SAMSN1 transcription may affect the progression and recurrence of GC, and therefore may represent a novel biomarker of GC.

Detection of serum melanoma‐associated antigen D4 in patients with squamous cell carcinoma of the esophagus

Despite improvements in surgical techniques, perioperative management, and multidisciplinary therapy, treatment outcomes of patients with esophageal squamous cell carcinoma (ESCC) remain poor. Therefore, development of novel molecular biomarkers, which either predict patient survival or become therapeutic targets, is urgently required. In the present study, to facilitate early detection of ESCC and predict its clinical course, we investigated the relationship of the serum level of melanoma-associated antigen (MAGE)-D4 to patients clinicopathological characteristics. Using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assays, we determined the levels of MAGE-D4 mRNA and protein in cell lysates and conditioned medium of cultures, respectively, of nine ESCC cell lines. Further, we determined MAGE-D4 levels in serum samples collected from 44 patients with ESCC who underwent radical esophagectomy without neoadjuvant therapy as well as from 40 healthy volunteers. Samples of conditioned medium and cell lysates contained comparable levels of MAGE-D4 that correlated closely with the levels of MAGE-D4 mRNA. Preoperative MAGE-D4 levels in the sera of 44 patients with ESCC, which varied from 0 to 2,354u2009pg/mL (314 ± 505u2009pg/mL, mean ± standard deviation), were significantly higher compared with those of healthy volunteers. By setting the cutoff at the highest value for healthy volunteers (50u2009pg/mL), the MAGE-D4-positive group of patients was more likely to have shorter disease-specific and disease-free survival compared with those of the MAGE-D4-negative group, although the differences were not statistically significant. Our results indicate that the elevation of preoperative serum MAGE-D4 levels in some patients with ESCC was possibly caused by excess production of MAGE-D4 by tumor cells followed by its release into the circulation. Clinical implications of serum MAGE-D4 levels should be validated in a large population of patients with ESCC.

Abstract 2909: Correlation between the expression of Sox2, Oct4 and deltaNp63 in clinical samples of resected esophageal cancers

Background. DeltaNp63 is the oncogenic isoform in several squamous cell carcinomas including esophageal cancer (EC), however, the mechanisms of regulation of the expression are still unclear. Sox2 and Oct4 are well known transcription factors which function in cancer stemness. We supposed that these two molecules work as transcription factors on deltaNp63, and verified the expression of these genes in cases of resected squamous cell EC. Methods. In 78 surgical specimens of squamous cell EC with no prior neoadjuvant therapy, messenger RNA (mRNA) expression of deltaNp63, Sox2 and Oct4 in EC tissues and corresponding normal tissues was analyzed by quantitative real-time reverse transcription-polymerase chain reaction assay. The mRNA expression was standardized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The expressions were compared in terms of clinicopathological factors and prognosis of postoperative survivals. Results. Peason correlation analysis showed significant correlations between expression of deltaNp63 and Sox2 in tumor tissues (r = 0.67, P Conclusions. Expression of deltaNp63 in EC tumor tissues showed positive correlation with expression of Sox2 and Oct4. Therefore, these two transcription factors may regulate the expression of deltaNp63. The deltaNp63 is not a strong prognostic factor, however, Oct4 might be a significant predictor for poor prognosis for overall survival in squamous cell EC. Citation Format: Yoshikuni Inokawa, Kenichi Inaoka, Fuminori Sonohara, Hisaharu Oya, Masamichi Hayashi, Yukiko Niwa, Naoki Iwata, Daisuke Kobayashi, Masahiko Koike, Yasuhiro Kodera, Shuji Nomoto. Correlation between the expression of Sox2, Oct4 and deltaNp63 in clinical samples of resected esophageal cancers. [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 2909.

Abstract 3423: Expression level of inflammasomes compornents NLRP3, NLRC4, and CASP1 in background non tumorous tissue were associated with worse prognosis for curatively resected hepatocellular carcinoma

Background and Objectives: When assessing hepatocellular carcinoma (HCC), it is quite important to examine prognostic factors in the background non tumorous liver tissue as well as HCC tissue itself. Inflammation has been thought to have some influence to malignancy of neoplasms and carcinogenesis. We focused on inflammasomes; multiprotein complex evoking key cascades of inflammation. Components of inflammasomes are investigated in mRNA level to identify molecular prognostic predictors for curatively resected HCC. Methods: Candidate genes that code pattern recognition receptors ; NLRP3, NLRC4, and AIM2 and caspase1 (CASP1) that mature IL-1β, IL-18 were investigated via real-time quantitative reverse transcription polymerase chain reaction in 158 consecutive curatively resected HCC cases at our department. We investigated each gene expression in both HCC tumor tissue (T) and background corresponding non tumorous tissue (CN) and super normal tissue (SN) taken from resected specimens of metastatic hepatic tumor was also assessed in this study. Statistical analyses were performed with Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate Cox proportional hazards models were used to determine the independent risk factors associated with the RFS and OS. Results: The expression level of NLRP3, NLRC4, and AIM2 (expression score/GAPDH×1000) were significantly higher in CN (NLRP3 median: 0.29 [range: 0.037-10.59], NLRC4 0.38[0.010-9.85] , AIM2 1.00 [0.016-43.62], n = 158 ) than in T (NLRP3 0.064 [0.0027-41.67] P Conclusions: Our findings suggested that NLRP3, NLRC4, and CASP1 expression in CN were significantly correlated with curatively resected HCC prognosis. Inflammation in background non tumorous tissue might be related with HCC malignancy and those were putative biomarker for curatively resected HCC. Citation Format: Fuminori Sonohara, Shuji Nomoto, Yoshikuni Inokawa, Mitsuro Kanda, Suguru Yamada, Tsutomu Fujii, Masahiko Koike, Hiroyuki Sugimoto, Michitaka Fujiwara, Yasuhiro Kodera. Expression level of inflammasomes compornents NLRP3, NLRC4, and CASP1 in background non tumorous tissue were associated with worse prognosis for curatively resected hepatocellular carcinoma. [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 3423. doi:10.1158/1538-7445.AM2015-3423