Tomoko Saito

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.

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.

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

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.

A temporal shift of the evolutionary principle shaping intratumor heterogeneity in colorectal cancer

Advanced colorectal cancer harbors extensive intratumor heterogeneity shaped by neutral evolution; however, intratumor heterogeneity in colorectal precancerous lesions has been poorly studied. We perform multiregion whole-exome sequencing on ten early colorectal tumors, which contained adenoma and carcinoma in situ. By comparing with sequencing data from advanced colorectal tumors, we show that the early tumors accumulate a higher proportion of subclonal driver mutations than the advanced tumors, which is highlighted by subclonal mutations in KRAS and APC. We also demonstrate that variant allele frequencies of subclonal mutations tend to be higher in early tumors, suggesting that the subclonal mutations are subject to selective sweep in early tumorigenesis while neutral evolution is dominant in advanced ones. This study establishes that the evolutionary principle underlying intratumor heterogeneity shifts from Darwinian to neutral evolution during colorectal tumor progression.Advanced colorectal cancers are characterised by intra-tumour heterogeneity dictated by neutral evolution. Here the authors analyse early colorectal tumours by whole-exome sequencing and find that Darwinian evolution determines the fate of early lesions in colorectal adenoma and carcinoma in situ.

Overexpression of FGFR1 Promotes Peritoneal Dissemination Via Epithelial-to-Mesenchymal Transition in Gastric Cancer

Background: Peritoneal dissemination (PD) is one of the most common causes of cancer-related mortality in gastric cancer (GC). We aimed to identify PD-associated genes and investigate their role in GC. Materials and Methods: We identified FGFR1 as a putative PD-associated gene using a bioinformatics approach. The biological significance of FGFR1 in epithelial-to-mesenchymal transition (EMT) was evaluated according to the correlation with genes that participated in EMT and FGFR1 knockdown experiments. The associations between FGFR1 expression and the clinicopathological features were examined. Results: FGFR1 expression positively correlated with SNAI1, VIM and ZEB1 expression, and negatively correlated with CDH1 expression. Knockdown of FGFR1 suppressed the malignant phenotype of GC cells. High FGFR1 expression significantly correlated with the peritoneal lavage cytology and synchronous PD positivity as well as poor prognosis. Conclusion: High FGFR1 expression was associated with PD via promotion of EMT and led to a poor prognosis of GC patients.

Cancer evolution and heterogeneity

Undoubtedly, intratumor heterogeneity (ITH) is one of the causes of the intractability of cancers. Recently, technological innovation in genomics has promoted studies on ITH in solid tumors and on the pattern and level of diversity, which varies among malignancies. We profiled the genome in multiple regions of nine colorectal cancer (CRC) cases. The most impressive finding was that in the late phase, a parental clone branched into numerous subclones. We found that minor mutations were dominant in advanced CRC named neutral evolution; that is, driver gene aberrations were observed with high proportion in the early‐acquired phase, but low in the late‐acquired phase. Then, we validated that neutral evolution could cause ITH in advanced CRC by super‐computational analysis. According to the clinical findings, we explored a branching evolutionary process model in cancer evolution, which assumes that each tumor cell has cellular automaton. According to the model, we verified factors to foster ITH with neutral evolution in advanced CRC. In this review, we introduce recent advances in the field of ITH including the general component of ITH, clonal selective factors that consolidate the evolutionary process, and a representative clinical application of ITH.