Shotaro Sakimura

Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green

We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

Decreased Expression of Fructose-1,6-bisphosphatase Associates with Glucose Metabolism and Tumor Progression in Hepatocellular Carcinoma

Fructose-1,6-bisphosphatase (FBP1), the rate-limiting enzyme in gluconeogenesis, is reduced in expression in certain cancers where it has been hypothesized to act as a tumor suppressor, including in hepatocellular carcinoma (HCC). Here, we report functional evidence supporting this hypothesis, providing a preclinical rationale to develop FBP1 as a therapeutic target for HCC treatment. Three independent cohorts totaling 594 cases of HCC were analyzed to address clinical significance. Lower FBP1 expression associated with advanced tumor stage, poor overall survival, and higher tumor recurrence rates. In HCC cell lines, where endogenous FBP1 expression is low, engineering its ectopic overexpression inhibited tumor growth and intracellular glucose uptake by reducing aerobic glycolysis. In patient specimens, promoter methylation and copy-number loss of FBP1 were independently associated with decreased FBP1 expression. Similarly, FBP1 downregulation in HCC cell lines was also associated with copy-number loss. HCC specimens exhibiting low expression of FBP1 had a highly malignant phenotype, including large tumor size, poor differentiation, impaired gluconeogenesis, and enhanced aerobic glycolysis. The effects of FBP1 expression on prognosis and glucose metabolism were confirmed by gene set enrichment analysis. Overall, our findings established that FBP1 downregulation in HCC contributed to tumor progression and poor prognosis by altering glucose metabolism, and they rationalize further study of FBP1 as a prognostic biomarker and therapeutic target in HCC patients. Cancer Res; 76(11); 3265-76. ©2016 AACR.

Somatic mutations in plasma cell-free DNA are diagnostic markers for esophageal squamous cell carcinoma recurrence

Objectives Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies owing to the high frequency of tumor recurrence. The identification of markers for early ESCC diagnosis and prediction of recurrence is expected to improve the long-term prognosis. Therefore, we searched for associations between tumor recurrence and cell-free DNA (cfDNA) mutations in blood plasma, which contains genetic markers for various cancer types. Experimental Design Genomic DNA from tumors and cfDNA from plasma were obtained from 13 patients undergoing treatment for newly diagnosed ESCC. Next-generation sequencing of cfDNA in plasma was performed to identify mutations in 53 cancer-related genes, in which recurrent mutations were previously detected in ESCC. cfDNA mutational profiles were compared before and after tumor resection in four patients. Furthermore, somatic mutations in serial plasma samples were monitored after treatment in four patients. Results We identified multiple concordant somatic mutations in cfDNA and primary tumor samples from 10 patients (83.3%) and in cfDNA and metastatic tumor samples from one patient (100%). Furthermore, the allele frequency of the concordant mutations in cfDNA changed concomitantly with tumor burden and increased approximately 6 months earlier than the detection of tumor recurrences by imaging tests in two patients. Conventional biomarkers, such as SCC and p53-Ab, did not reflect tumor recurrences. Conclusions The present multigene panel, which enabled the diagnosis of tumor recurrence with greater accuracy than did using standard tumor markers or imaging methods, is expected to greatly facilitate standard, postoperative follow-up monitoring in ESCC.

An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma.

Background Few driver genes have been well established in esophageal squamous cell carcinoma (ESCC). Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes. Methods We searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort. Results We found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC. Conclusion Our integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.

Loss of CDCP1 expression promotes invasiveness and poor prognosis in esophageal squamous cell carcinoma.

BackgroundHuman CDCP1 gene, located on chromosome 3p21.3, is a transmembrane glycoprotein widely expressed in epithelial tissues, and its role in cancer remains to be understood.MethodsUsing microarray profiles of gene expression and copy number data from 69 esophageal squamous cell carcinoma (ESCC) samples, we performed informatics analyses to reveal the significance of CDCP1 expression. We also performed migration and invasion assays of siRNA-targeted CDCP1-transfected cells and CDCP1-overexpressing cell in vitro. Moreover, we evaluated the clinical magnitude of CDCP1 expression in esophageal squamous cell cancer cases.ResultsAllelic loss of chromosome 3p was confirmed by copy number analysis. The expression level of CDCP1 in tumor tissue was significantly lower than that in corresponding normal tissue. siRNA targeting of CDCP1 promoted the migratory and invasive abilities of esophageal cancer cell lines, whereas both abilities were reduced in CDCP1-overexpressing cells. Gene set enrichment analysis showed that expression levels of CDCP1 were associated with tumor differentiation and metastasis, consistent with the result of clinicopathologic analyses. Finally, multivariate analysis revealed that the expression level of CDCP1 was an independent prognostic factor for survival.ConclusionsLoss of CDCP1 expression may be a novel indicator for biological aggressiveness in ESCC.

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.

Rapid diagnosis of lymph node metastasis in breast cancer using a new fluorescent method with γ-glutamyl hydroxymethyl rhodamine green.

Sentinel lymph node biopsy is performed as a standard procedure in breast cancer surgery, and the development of quick and simple methods to detect metastatic lesions is in high demand. Here, we validated a new fluorescent method using γ-glutamyl hydroxymethyl rhodamine green to diagnose metastatic lymph nodes in breast cancer. One hundred and forty-nine lymph nodes from 38 breast cancer patients were evaluated in this study. Comparison of fluorescent and pathological images showed that this fluorescent method was successful for visualizing breast cancer cells in lymph nodes. This method had a sufficiently high sensitivity (97%), specificity (79%) and negative predictive value (99%) to render it useful for an intraoperative diagnosis of cancer. These preliminary findings suggest that this novel method is useful for distinguishing non-cancerous specimens from those in need of careful examination and could help save time and cost for surgeons and pathologists.

Altered Expression of Hippo Signaling Pathway Molecules in Intrahepatic Cholangiocarcinoma

Objective: MOB1, a core component of the Hippo signaling pathway, suppresses cell proliferation, and MOB1 liver conditional knockout mice develop intrahepatic cholangiocarcinoma (ICC). However, its clinical significance in human ICC has not been established. The aim of this study was to characterize protein levels and the role of Hippo and TGF pathways in ICCs. Methods: The protein levels of yes-associated protein 1 (YAP1), MOB1, Smad2, and TGFβ2 in 88 ICC cases were analyzed. Protein level was graded by a scoring system; then, the clinicopathological factors, including prognosis, were analyzed based on protein level. Results: Nuclear overexpression of YAP1 was seen in 28 cases (31.8%), and it was significantly associated with a poor overall survival rate (p = 0.01). MOB1 expression decreased in 42 cases (47.7%) and was associated with a poor overall survival rate (p = 0.02). SMAD2 nuclear localization was significantly correlated with a high YAP1 level independent of TGFβ2. Multivariate analysis revealed that a high YAP1 level, a low MOB1 level, and lymphatic permeation were independent risk factors for overall survival. Conclusions: These results showed that key components of the Hippo signaling pathway are aberrantly expressed and associated with the malignant potential of human ICC.

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.

Abstract 5169: Omics approach to identify driver genes for peritoneal dissemination of gastric cancer cells

Background Little is known about the molecular mechanisms of peritoneal dissemination in gastric cancer cells. In this study, to clear the molecular mechanisms of accelerating the peritoneal dissemination, we performed to identify significant genes which give rise to peritoneal dissemination and poor prognosis of gastric cancer. We conducted expression array analysis in mice model of peritoneal dissemination and in 200 cases of gastric cancer cases, and compared with each other to identify driver genes. Methods As described previously, three parental scirrhous gastric carcinoma-derived cell lines (HSC-39, HSC-44PE and HSC-58) were established from human scirrhous gastric carcinoma. Moreover, inoculating each parental cell lines into the gastric wall of nude mice, four matched highly peritoneal-metastatic cell lines (HSC-39As8, HSC-44As3, HSC-58As1 and HSC-58As9) were established after 12 cycles of stepwise selection. To identify gene expression signatures for peritoneal dissemination, we performed expression analysis for matched parental and each resultant cell line. Next, to examine the relationship between gene expression signatures and clinical behaviors, we performed synthetic analysis of gene expression signatures from in vivo peritoneal dissemination mouse model data and expression data set containing 200 gastric tumors from the Singapore cohort. Results (1) Our synthetic analysis, EEM analysis, which tested the functionality of the input genes sets on the basis of their coherence, showed that one gene set from the 58As9 cell line harbored a coherently expressed subset of significant size. (2) We found 57 genes in the coherent subset which had clinical correlation. (3) We found that peritoneal metastases patients tended to show higher expression of such genes, and patients prognosis with higher expression of such genes tended to be poor. (4) Among such genes, we focused on chemokine(C-X-C motif) receptor 7(CXCR7). CXCR7 expression was significantly higher in 58As9 than in HSC58(P Conclusions In conclusion, synthetic analysis from two data sets indicated several candidate driver genes for strongly accelerating gastric cancer peritoneal dissemination, the current approach to compare gene signatures in vivo and clinical samples with accurate clinical annotations enabled us to identify brand-new driver candidate genes. Among them, CXCR7 might play an important role in peritoneal dissemination and CXCR7 might be a therapeutic target for suppression of gastric cancer peritoneal dissemination in the near future. Note: This abstract was not presented at the meeting. Citation Format: Sho Nambara, Junji Kurashige, Tomoko Saito, Hisateru Komatsu, Masami Ueda, Shotaro Sakimura, Hidenari Hirata, Ryutaro Uchi, Yuki Takano, Yoshiaki Shinden, Tomohiro Iguchi, Hidetoshi Eguchi, Keishi Sugimachi, Yoshihiko Maehara, Koshi Mimori. Omics approach to identify driver genes for peritoneal dissemination of gastric cancer cells. [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 5169. doi:10.1158/1538-7445.AM2015-5169