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Isotopomer distribution analysis using [U-13C]glucose in HuH7 cells with or without ectopic FBP1 expression.

Promoter methylation and copy number profiles of FBP1 in liver cancer cell lines from the Cancer Cell Line Encyclopedia.

Overall survival based on the expression levels of genes specific to gluconeogenesis and aerobic glycolysis.

The expression levels of genes specific to gluconeogenesis in HCC cases.

Supplementary Materials and Methods, and Legends for Supplementary Figures.

FBP1 expression and mutational profiles across 234 HCC cases from The Cancer Genome Atlas.

The activity of gluconeogenesis and aerobic glycolysis predicted survival in patients with HCC.

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

Background: Long non-coding RNA (lncRNA) is known to play important roles in proliferation or metastasis in several human cancers. A recent study reported that lncRNA activated by TGF-β (lncRNA-ATB) induced Epithelial-Mesenchymal Transition (EMT) through the TGF-β/miR-200s/ZEB axis in hepatocellular carcinoma (HCC), therefore the high lncRNA-ATB patients in HCC had poor prognosis. EMT through the TGF-β/miR-200s/ZEB axis reported to be involved in invasion, metastasis, or dessemination in gastric cancer. Therefore, we focused on the clinical significance of lncRNA-ATB expression in gastric cancer. Materials and Methods: qRT-PCR was performed to examine expression of lncRNA-ATB, miR-200b, and miR-200c in gastric cancer tissues (n = 183). Then we divided patients into high and low lncRNA-ATB expression groups using lncRNA-ATB/GAPDH ≥0.55 or genetic alterations, such as lncRNA-ATB, miR-200s, and ZEB1 levels, with respect to the EMT phenotype. Results: LncRNA-ATB expression levels in gastric cancer patients weren't associated with the clinicopathological factors, gender, age, tumor size, histological type, depth of tumor invasion, lymph node metastasis, lymphatic invasion, vascular invasion, liver metastasis, peritoneal dissemination, or distant metastasis. However, the high lncRNA-ATB group experienced a lower 5-year survival rate compared to the low lncRNA-ATB group (P = 0.003), and multivariate analysis indicated that lncRNA-ATB was an independent prognostic factor (HR = 3.18; 95%CI: 1.47-7.29; P = 0.0031). The lncRNA-ATB level was inversely associated with miR-200s in gastric cancer cell lines. The inverse association was validated in miR-200c and lncRNA-ATB in gastric cancer patients. Moreover, lncRNA-ATB and ZEB1 levels increased and miR-200c levels reduced in low lncRNA-ATB cells treated with TGF-β1 after 10 d of treatment. Morphologies also changed from epithelial to mesenchymal form. Conclusions: LncRNA-ATB plays an important role in EMT to promote invasion and metastasis through the TGF-β/miR-200s/ZEB axis, resulting in a poor prognosis in gastric cancer. Further investigations will demonstrate the significance of lncRNA-ATB as a novel lncRNA biomarker indicative of a poor prognosis in gastric cancer patients. Citation Format: Tomoko Saito, Junji Kurashige, Sho Nambara, Hisateru Komatsu, Hidenari Hirata, Masami Ueda, Shotaro Sakimura, Ryutaro Uchi, Yuki Takano, Yoshiaki Shinden, Tomohiro Iguchi, Hidetoshi Eguchi, Keishi Sugimachi, Kazunari Murakami, Koshi Mimori. A long non-coding RNA activated by TGF-β can predict the prognosis of gastric cancer patients. [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 143. doi:10.1158/1538-7445.AM2015-143

Introduction Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the third leading cause of all cancer deaths worldwide. A fraction of HCC has increased activity of Wnt/β-catenin (Wnt) pathway, which reportedly enhances malignant phenotypes like epithelial-mesenchymal transition (EMT) in various kinds of cancer. Identification of Wnt pathway target and regulator genes gives us better understanding of molecular biological behavior of HCC. In this study, we performed in silico screening for a novel Wnt pathway target and regulator genes in human HCC using public databases. Method First, we obtained TCGA data sets of human HCC including mRNA expression (268 samples) and mutational status (198 samples) from the Broad GDAC Firehose web site (gdac.broadinstitute.org/). Next, for β-catenin transcriptional target genes prepared by literature curation, we performed EEM analysis (Niida et al. 2009) on the HCC expression data set to obtain Wnt pathway target genes in HCC. Assuming the sum of mRNA expression of the Wnt pathway target genes as Wnt pathway activity, we then measured association between the pathway alteration and the activity by linear multiple regression analysis ([Wnt pathway activity] = a0 + a1[CTNNB1 mutation] + a2[AXIN1 mutation] + a3[AXIN2 mutation]+a4[APC mutation]+ a5[CTNNB1 mRNA expression] : a0∼a5; partial regression coefficient). By focusing on known epithelial markers (CDH1, KRT18, MUC1, OCLN, TJP1) and mesenchymal markers (ACTA2, CDH2, FN1, VIM, VTN), we also examined association between the Wnt pathway activity and EMT-manifesting genotype. Finally, we performed multiple regression analysis to search for novel Wnt pathway regulator genes ([Wnt pathway activity] = b0 + b1[CTNNB1 mutation] + b2[APC mutation]+ b3[CTNNB1 mRNA expression] + b4[mRNA of a regulator] : b0∼b4; partial regression coefficient). Result (1)EEM identified AASS, ASPSCR1, BMP4, C6orf97, DUT, GGH, GNPAT, GREB1, HGD, IKBKAP, ITPR2, LAMA3, LGR5, NEK3, RELN, RHBG, RHOBTB1, SLC13A3, SMYD2, TBX3, TDGF1, TRIB2, VSNL1, ZBTB38 as the Wnt pathway target genes in HCC. (2)Multiple regression analysis revealed the Wnt pathway activity had strong correlations with CTNNB1 mutation (p-value < 2.00×10-16), APC mutation (p-value = 2.73×10-3), and CTNNB1 expression (p-value = 2.19×10-07) (3)The Wnt pathway activity had a strong correlation with the EMT genotype (p-value = 3.58×10-4). (4)We identified 100 statistically significant Wnt pathway regulator candidates (q-values < 1.00×10-10). Conclusion Our in silico screening identified novel Wnt pathway target genes in HCC, whose expression profiles were correlated with Wnt pathway alterations and the EMT-manifesting gene signature. We also have predicted Wnt pathway regulator genes, and are now conducting in vitro validation. Citation Format: Hisateru Komatsu, Atsushi Niida, Masami Ueda, Hidenari Hirata, Ryutaro Uchi, Sho Nambara, Tomoko Saito, Shotaro Sakimura, Yuki Takano, Yoshiaki Shinden, Tomohiro Iguchi, Hidetoshi Eguchi, Keishi Sugimachi, Yuichiro Doki, Masaki Mori, Koshi Mimori. In silico screening for novel Wnt/β-catenin pathway target and regulator genes in human 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 1948. doi:10.1158/1538-7445.AM2015-1948

Method After spraying with gGlu-HMRG reagent to the samples, we took images within 5 minutes and analyzed the fluorescence intensity of them. Comparing the changes of fluorescence intensity and pathological evaluations, we examined how this fluorescent method could reflect the pathological status in resected breast tissues (n = 108 from 36 cases), margins of breast conserving surgery specimens (n = 7 from 5 cases) and axillary lymph nodes (n = 149 from 38 cases). Result 1. The sensitivity and specificity were 92% and 94% respectively for binary classification (normal / abnormal) of various breast tissues. 2. We could detect all malignant lesions in the surface of surgical margins of breast conserving surgery specimens. Additionally, we could detect large tumor-free lesions as fluorescent negative regions. 3. The sensitivity and specificity were 97% and 79% respectively for diagnosing metastatic and non-metastatic axillary lymph nodes. Discussion and conclusion It was well feasible to distinguish breast tumor tissues from normal surrounding tissues by this new method. Since it is simple and non-tissue-destructive, it could be readily adopted in the clinical setting. We anticipate that it will reduce costs and the burden on histopathology services in the intraoperative pathological diagnosis of breast conserving surgery and sentinel lymph node biopsy. This technical innovation enable us to visualize tumor cells in the body intraoperatively and evolve the cancer surgery that needs the complete resection of cancer cells. *1 Urano, Y. et al. Rapid cancer detection by topically spraying a γ-glutamyltranspeptidase-activated fluorescent probe. Sci. Trans. Med. 3, 110ra119 (2011). Citation Format: Yoshiaki Shinden, Hiroki Ueo, Taro Tobo, Ayako Ganachi, Hisateru Komatsu, Sho Nambara, Tomoko Saito, Masami Ueda, Hidenari Hirata, Shotaro Sakimura, Yuki Takano, Ryutaro Uchi, Tomohiro Iguchi, Hidetoshi Eguchi, Keishi Sugimachi, Yoko Kubota, Yuichiro Kai, Yuko Kijima, Shoji Natsugoe, Hiraki Ueo, Yasuteru Urano, Koshi Mimori. The new and rapid technique of detecting breast cancer cells using new fluorescent probe ‘gGlu-HMRG’ and its clinical application. [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 218. doi:10.1158/1538-7445.AM2015-218

Backgrounds/Aims Most gastric cancer cells acquire epithelial-mesenchymal transition (EMT) to promote malignant cells to form metastasis in gastric cancer cases. Recent studies proved the roles of microRNAs on the regulation of EMT through suppressing EMT-related transcription factors. Of those, miR-506 inhibits expression of SNAI2 and PRRX1 and aberrant low expression of miR-506 relates to poor prognosis in ovarian and breast cancers. The aim of this study is to investigate the role of miR-506 on survival and EMT in Japanese gastric cancer (GC) patients. Methods 144 patients with surgically resected primary GC were included in this study. MicroRNA and mRNA were measured using real-time PCR analysis. Clinicopathological factors including prognosis that were dependent on miR-506 expression were analyzed. In vitro, we established miR-506 overexpressing human GC cell lines (MKN-7 and MKN-45). The proliferation, migration and luciferase assay of 3′UTR of SNAI2 were performed. Results (1) The low expression of miR-506 was significantly correlated with poor overall survival in the univariate model (log-rank p = 0.02 ) and multivariate model (hazard ratio = 1.78, p = 0.05). The miR-506 expression was inversely correlated with SNAI2 expression (p = 0.01). The low expression of miR-506 was significantly correlated with histologically poorer differentiation that showed more invasive properties (p = 0.02). (2) In miR-506 overexpressed GC cell lines, miR-506 suppressed the SNAI2 expression by binding to 3′UTR of SNAI2 gene, and resulted in the increased expression of E-cadherin. MiR-506 overexpression significantly suppressed the cell proliferation and the cell migration compared to the miR-control transfected cells. Conclusions This study showed that miR-506 directly controlled EMT by regulating SNAI2, and was a independent prognostic factor in Japanese GC patients. Our data indicated that miR-506 could be a candidate of EMT inhibitor in GC patients. Univariate and multivariate analysis of clinicopathological features for 5-year overall survivalUnivariate analysisMultivariate analysisFeaturesHR95%CIP valueHR95%CIP valueAge (>70/70≥)0.6260.345-1.0890.099---Gender (male/female)1.4910.844-2.7810.174---Histological grade(Well & Moderately/Poorly & others)1.6470.959-2.8920.071Depth(T1, 2/T3, 4)4.9572.389-12.040 Citation Format: Shotaro Sakimura, Junji Kurashige, Keishi Sugimachi, Masami Ueda, Hidenari Hirata, Yoshiaki Shinden, Etsuko Sakimura, Tae Matsumura, Yuki Takano, Ryutaro Uchi, Hiroki Ueo, Hidetoshi Eguchi, Tomoya Sudo, Sumio Hoka, Koshi Mimori. Decreased expression of miR-506 induced epithelial-mesenchymal transition and poor prognosis in gastric cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1478. doi:10.1158/1538-7445.AM2014-1478

Background PLS3 gene, located on chromosome Xq23, encodes a protein 10 product (PLS3) that reduces F-actin disassembly and inhibits cofilin-mediated depolymerization of actin fibers. We previously reported that PLS3 is a novel marker for circulating tumor cells in colorectal cancer (CRC), and the high expression of PLS3 in tumor tissue is associated with a poor prognosis in patients with primary CRC. In this study, we investigated its role in functional significance for metastatic potential of CRC performing in vitro and in vivo studies. Method Four patients with surgically resected both primary CRC and liver metastasis were included in this study. Those tissues were performed with the PLS3-immunohistochemical examination (PLS3-IHC). In vitro, we cultured LOVO, DLD-1 and HCT116 which caused no liver metastasis by injecting them into the spleen of NOD/SCID mice. We stably enhanced the expression of PLS3 in the cells by transfecting the lentiviral vector expressing PLS3. The expression levels of EMT-related genes were examined with real time RT-PCR. The migration, invasion and sphere formation assay were also performed. In vivo, NOD/SCID mice were divided into two groups, which included mice transplanted with only PLS3-transfected cells and mice transplanted with PLS3-transfected and PLS3-attenuated cells together, by injecting into the spleen. We calculated the established primary splenic tumors and liver metastasis. PLS3-IHC was also performed to the sacrificed tissues. Result (1) In the PLS3-IHC of four CRC samples, all the liver metastasis tissues included stained areas, but only one primary tissue showed PLS3-staining. This was the evidence that cancer cells expressing PLS3 had metastatic properties. (2) PLS3-transfected cells had the significant decrease of E-cadherin, while the expression levels of TWIST, ZEB and vimentin were significantly higher in PLS3-transfected than PLS3-attenuated cells. PLS3-transfected LOVO significantly induced invasion and migration, compared to PLS3-attenuatd LOVO. In the sphere formation assay, the high expression of PLS3 significantly increased the proportion of sphere generating cells (DLD-1) (p (3) In vivo, PLS3-transfected cells (DLD-1 and HCT116) significantly induced the growth of liver metastasis. The volumes of liver metastasis of mice transplanted with the mixtures were significantly smaller than those of only PLS3-transfected cells. The liver metastasis tissues showed only stained areas in both groups in PLS3-IHC. However, the established splenic tumor of mice transplanted with the mixtures displayed not only the stained but also the nonstained areas in PLS3-IHC. Conclusion In conclusion, our study suggests experimental evidence to support the functional significance of PLS3 to induce epithelial-mesenchymal trandition, stemness and liver metastasis of human CRC. Citation Format: Masami Ueda, Keishi Sugimachi, Junji Kurashige, Shotarou Sakimura, Hidenari Hirata, Ryutarou Uchi, Yuki Takano, Hiroki Ueo, Tae Matsumura, Yoshiaki Shinden, Hidetoshi Eguchi, Tomoya Sudo, Masaki Mori, Koshi Mimori. Aberrant expression of Plastin3 (PLS3) induces liver metastasis via enhancing the epithelial-mesenchymal transition and stemness in colorectal cancer (CRC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1138. doi:10.1158/1538-7445.AM2014-1138

Background: Recent studies have been focused on the relationship between cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT). Unlike classical EMT inducers, PRRX1 is involved in the acquisition of CSC-like properties when its down-regulation induces mesenchymal-epithelial transition in breast cancer. Meanwhile, hepatocellular carcinoma (HCC) is one of the most intractable malignancies, and it is required to identify a bona-fide molecular target to regulate tumor progression and the treatment resistance. The purpose of this study is to investigate the role of PRRX1 expression on clinical significance and CSC-like properties in HCC. Methods: (1) PRRX1 expression was analyzed in 62 resected primary HCC cases by quantitative RT-PCR (qRT-PCR). Clinicopathological factors and overall survival (OS) according to PRRX1 expression were analyzed. (2) The gene set enrichment analysis (GSEA) on the published clinical data set of 242 HCC samples (GSE14520) was applied to investigate the association of PRRX1 expression levels and the stem cell-like properties. (3) PRRX1 stably expressing HCC cell lines (HuH7) were established using a lentiviral vector. The expression levels of the hepatic CSC surface markers (CD13, CD133 and EpCAM) were analyzed by flowcytometry and qRT-PCR. Furthermore, the ability of sphere formation and in vitro chemosensitivity to 5-FU were evaluated to assess the CSC-like properties. Results: (1) Univariate analysis showed that 5-year OS of the low PRRX1 expression group was significantly shorter than that of the high PRRX1 expression group (49.8% vs. 80.3%, P=0.014). On multivariate analysis, low PRRX1 expression was an independent prognostic factor for HCC (P=0.026). (2) GSEA analysis on the 247 HCC data set indicated that the down-regulation of PRRX1 was significantly correlated with the stem cell signature (P=0.039). Furthermore, using gene sets of polycomb repressive complex 2 (PCR2) targets and H3 lysine-27 trimethylation mediated by PCR2, HCC with low PRRX1 expression showed parallel enrichment patterns with human embryonic stem cells (P=0.002 and P Conclusions: The down-regulation of PRRX1 expression contributes to the poor prognosis through the acquisition of CSC-like properties in HCC patients. We might apply the aberrant expression of PRRX1 as a novel clinical biomarker for prediction of the malignant potential in HCC cases. Citation Format: Hidenari Hirata, Keishi Sugimachi, Ryutaro Uchi, Junji Kurashige, Tae Matsumura, Yuki Takano, Hiroki Ueo, Masami Ueda, Shotaro Sakimura, Yoshiaki Shinden, Hidetoshi Eguchi, Tomoya Sudo, Masakazu Hirakawa, Hiroshi Honda, Koshi Mimori. Downregulation of PRRX1 confers cancer stem cell-like properties and poor prognosis in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3882. doi:10.1158/1538-7445.AM2014-3882