Takeshi Toyozumi

Histone Demethylase LSD1 Inhibitors Prevent Cell Growth by Regulating Gene Expression in Esophageal Squamous Cell Carcinoma Cells.

BackgroundThe expression of genes can be influenced by the balance of histone acetylation and/or histone demethylation, with an imbalance of these processes possibly observed in many cancers. The histone demethylase LSD1 inhibitor activity is associated with selective transcriptional regulation and alterations in the gene expression. However, the exact mechanisms underlying the antitumor effects of LSD1 inhibitors are not fully understood.MethodsThe antitumor effects of NCL1, an LSD1 inhibitor, in esophageal squamous cell cancer (ESCC) cell lines were evaluated. A comprehensive analysis of the changes in the gene expression in ESCC cell lines induced by NCL1 was carried out using a microarray analysis. A loss-of-function assay using a siRNA analysis was performed to examine the oncogenic function of the gene.ResultsNCL1 strongly inhibited the cell growth of T.Tn and TE2 ESCC cells and induced apoptosis. According to the microarray analysis, 81 genes in the T.Tn cells and 149 genes in the TE2 cells were up- or down-regulated 2-fold or more by NCL1 exposure. Among these genes, 27 were contained in both cell lines and exhibited similar expression patterns. PHLDB2, one of the genes down-regulated by NCL1, was overexpressed in the ESCC tumor tissues. Moreover, a high expression level of PHLDB2 was found to be significantly correlated with poor prognosis.ConclusionsThe present observations of the comprehensive analysis of the gene expression levels provide insight into the mechanisms underlying the antitumor effects of LSD1 inhibitors in ESCC patients.

Usefulness of microRNA‑375 as a prognostic and therapeutic tool in esophageal squamous cell carcinoma.

The aim of this study was to clarify the importance of microRNA‑375 (miR‑375) expression in patients with esophageal squamous cell carcinoma (ESCC) and to examine the in vivo antitumor effects of miR‑375 in a model of ESCC using a non‑viral delivery system. We estimated the miR‑375 and LDHB and AEG‑1/MTDH mRNA expression of the ESCC tumors from 85 patients. The correlation between the miR‑375 expression and clinicopathological features, including the prognosis, were evaluated. The presence of high miR‑375 expression was associated with lymphatic vessel invasion, while a low expression of miR‑375 significantly correlated with a poor prognosis for the 85 ESCC patients. We also found that there was a significant inverse correlation between the expression of miR‑375 and that of LDHB. Before the examination of miR‑375 in the in vivo assay, we confirmed that atelocollagen prolonged the accumulation of miRNA by using fluorescently‑labeled miRNA and an in vivo imaging system. We injected the miR‑375/atelocollagen complex or a control‑miRNA/atelocollagen complex into mice bearing TE2 and T.Tn xenografts via subcutaneous (s.c.) injections. The growth of both the TE2 and T.Tn tumors in the miR‑375 groups was significantly suppressed compared with that in the control‑miRNA groups. In addition, The LDHB mRNA expression of TE2 xenografts was significantly downregulated after miR‑375 treatment. In conclusion, it might be possible for the level of miR‑375 expression to be a utilized as a prognostic indicator for ESCC patients. The administration of miR‑375 using a non‑viral delivery might represent a powerful new treatment for ESCC.

Suture Granuloma With False-Positive Findings on FDG-PET/CT Resected via Laparoscopic Surgery

A 61-year-old woman who had undergone total hysterectomy 16 years previously exhibited a pelvic tumor on computed tomography (CT). F-18 fluorodeoxyglucose (FDG) combined positron emission tomography (PET)/CT imaging revealed a solitary small focus of increased FDG activity in the pelvis. A gastrointestinal stromal tumor originating in the small intestine or another type of tumor originating in the mesentery (desmoid, schwannoma, or foreign body granuloma) was suspected; therefore, laparoscopic resection was conducted. A white, hard tumor was found to originate from the mesentery of the sigmoid colon and adhered slightly to the small intestine. The tumor was resected with a negative margin, and the pathologic diagnosis was suture granuloma. The possibility of suture granuloma should be kept in mind in cases of tumors with positive PET findings and a history of surgery close to the lesion. However, it is difficult to preoperatively diagnose pelvic tumors using a biopsy. Therefore, considering the possibility of malignancy, it is necessary to achieve complete resection without exposing the tumor.

A 3-step gradual dilation method: a new safe technique of percutaneous endoscopic gastrostomy for obstructive esophageal cancer.

Although percutaneous endoscopic gastrostomy (PEG) is the preferred method to provide enteral nutrition for a longer time period, in obstructive esophageal cancer, we cannot safely perform endoscopic access to the stomach even with the ultrathin endoscope. We experienced 1 fatal case due to esophageal perforation caused by balloon dilation, and hence, we developed a safer method. We treated 4 patients with obstructive esophageal cancer using a 3-step gradual dilation method with nasogastric tubes (from 8 to 16 Fr). After about 2 weeks of initial dilation, we could safely perform endoscopic access to the stomach with the ultrathin endoscope and PEG placement using the introducer technique. The 3-step gradual dilation method is a safe and easy procedure for endoscopic access to the stomach. It can be used to provide enteral access as a palliative treatment for patients with obstructive esophageal cancer that is not suitable for conventional PEG placement.

Screening of Alternative Drugs to the Tumor Suppressor miR-375 in Esophageal Squamous Cell Carcinoma Using the Connectivity Map

Objective: The aim of this study was to identify alternative compounds to the tumor suppressor miR-375 using the connectivity map (CMAP) and to validate the antitumor effects of the identified drugs in esophageal squamous cell carcinoma (ESCC). Methods: Gene profiling of miR-375-treated TE2 and T.Tn cells was applied in order to search the CMAP database. Among the compounds identified using the CMAP, we focused on 8 drugs [(-)-epigallocatechin-3-gallate, metformin, rosiglitazone among others], excluding 2 drugs among the top 10 compounds. We evaluated whether these compounds possess tumor-suppressive functions in ESCC. Results: A cytotoxicity assay showed that the sensitivity of TE2 and T.Tn cells treated with the 8 compounds was evaluated based on IC50 values of 42.9 µM to 3.8 mM. A cell cycle analysis revealed that the percentage of TE2 and T.Tn cells incubated with 6 compounds in the G0/G1 phase or the G2/M phase increased by approximately 40-80%. A TUNEL assay showed that the percentages of apoptotic cells treated with almost all compounds were significantly increased (p < 0.05) compared with the control cells. Conclusion: The CMAP database is a useful tool for identifying compounds affecting the same molecular pathways, particularly products that are difficult to apply via practical approaches, such as microRNAs.

Antitumor effects of metformin are a result of inhibiting nuclear factor kappa B nuclear translocation in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is an intractable digestive organ cancer that has proven difficult to treat despite multidisciplinary therapy, and a new treatment strategy is demanded. Metformin is used for type 2 diabetes mellitus and its antitumor effects have been reported recently. Metformin exerts antitumor effects in various respects, such as inhibiting inflammation, tumor growth and epithelial‐mesenchymal transition (EMT). However, few reports have described the efficacy of metformin on ESCC, and their findings have been controversial. We analyzed the antitumor effects of metformin and clarified its effects on anti‐inflammation, growth suppression and EMT inhibition. Activation of nuclear factor kappa B (NF‐κB), the major transcription factor induced by inflammation, was investigated by immunostaining. We found that localization of NF‐κB in the nucleus was reduced after metformin treatment. This suggests that metformin inhibited the activation of NF‐κB. Metformin inhibited tumor growth and induced apoptosis in ESCC cell lines. Associated with EMT, we examined cell motility by a wound healing assay and the epithelial marker E‐cadherin expression of various ESCC cell lines by western blotting. Metformin inhibited cell motility and induced E‐cadherin expression. In conclusion, metformin showed multiple antitumor effects such as growth suppression, invasion inhibition, and control of EMT by inhibiting NF‐κB localization on ESCC. Further exploration of the marker of treatment efficacy and combination therapy could result in the possibility for novel treatment to use metformin on ESCC.

ZNF750 Expression Is a Potential Prognostic Biomarker in Esophageal Squamous Cell Carcinoma

Objective: ZNF750, a transcriptional regulator of epidermal differentiation, has been identified as a tumor suppressor in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to investigate the clinical and prognostic significance of ZNF750 expression and to evaluate the effect of ZNF750 knockdown on cell proliferation, migration, and invasion in ESCC. Methods: A total of 124 patients with ESCC who underwent curative esophagectomy were evaluated in this study. The expression of ZNF750 in surgical specimens was immunohistochemically assessed and used in the analysis of clinicopathological features and overall survival (OS). The molecular role of ZNF750 was investigated by ZNF750 knockdown using small interfering RNA (siRNA) in ESCC cell lines. Results: Low ZNF750 expression had a significant correlation with positive lymph node metastasis (p = 0.028). Furthermore, there was a significant relationship between low expression of ZNF750 in ESCC and a poor OS, and a multivariate analysis showed that low ZNF750 expression was an independent prognostic factor (p = 0.020). The cell growth, migration, and invasion were significantly increased by downregulation of ZNF750. Conclusions: The low expression of ZNF750 was significantly associated with a poor prognosis, and ZNF750 expression may, therefore, be a reliable prognostic biomarker in ESCC.

Fra-1 Regulates the Expression of HMGA1, Which is Associated with a Poor Prognosis in Human Esophageal Squamous Cell Carcinoma

BackgroundThe expression of Fos-related antigen 1 (Fra-1) affects tumor progression, migration, and invasion. In this study, we identified the genes regulated by Fra-1 in esophageal squamous cell carcinoma (ESCC).MethodsWe constructed Fra-1 knockdown models via the transfection of small interfering RNA (siRNA) into ESCC cell lines (TE10, TE11). The expression levels of the genes in the knockdown models were analyzed using a microarray and a Biobase Upstream Analysis, while the expression levels of the candidate genes in the primary tumors of surgical specimens obtained from ESCC patients were determined using real-time polymerase chain reaction (PCR) and immunohistochemical staining. The clinicopathological features were then analyzed.ResultsThe Biobase Upstream Analysis showed the high-mobility-group protein-1 (HMGA1) to be a significant gene regulated by Fra-1. Actual binding of Fra-1 to the promotor region of HMGA1 was revealed in subsequent chromatin immunoprecipitation PCR experiments. Patients with a positive HMGA1 expression had a poor prognosis, and a multivariate analysis demonstrated a positive HMGA1 expression to be a significant independent prognostic factor.ConclusionHMGA1 is regulated by Fra-1 in ESCC, and the HMGA1 expression is significantly associated with a poor prognosis in ESCC patients. Downregulation of the HMGA1 expression may become a practical treatment strategy against ESCC in the future.

Abstract 4032: Functional analysis of exosomes derived from human esophageal squamous cell carcinoma

Exosomes are small membrane vesicles (30-100 nm) released by many types of cells including cancer cells, and are thought to play an important role in cell-to-cell communications. Exosomes contain lipids, proteins, and nucleic acids such as mRNAs or microRNAs, they are tought to have relationship with tumor growth or metastasis. Our laboratory was previously reported the level of microRNA-1246, expressed mainly in tumor tissues of esophageal squamous cell carcinoma (ESCC), was significantly high in exosomes extracted from the serum of ESCC patients. However, the exosome dynamics and transportation between cancer cells and the other cells are still unclear. In this study, we assessed the cell-to-cell communication via exosomes with fluorescent imaging and analyzed the effect of cancer cell derived exosome to tumor progression. In order to image and analyze the movement of cancer cell-derived exosomes, green fluorescent protein (GFP)-tagged CD63, which is a general marker of exosomes, was expressed in human esophageal squamous cancer cell line TE2. Exosomes were extracted from supernatant with ultracentrifuge method. Extracted GFP-tagged exosomes were added to the other cells to obtain the images of transportation. The effects of exosomes to tumor progression were also assessed with cell proliferation assay, invasion oassay, and migration assay. Our data indicates that cancer cell derived exosomes affect tumor progression. Citation Format: Yasunori Matsumoto, Masayuki Kano, Yasunori Akutsu, Takanori Nishimori, Naoyuki Hanari, Isamu Hoshino, Kentaro Murakami, Takeshi Toyozumi, Hiroshi Suito, Masahiko Takahashi, Nobufumi Sekino, Yoshihiro Nabeya, Hisahiro Matsubara. Functional analysis of exosomes derived from human esophageal squamous cell 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 4032. doi:10.1158/1538-7445.AM2015-4032

Contents Vol. 87, 2014

A.B. Benson, Chicago, Ill. A. Chang, Singapore A.L. Cheng, Taipei J.F. Cleary, Madison, Wisc. M. Dietel, Berlin M.S. Ernstoff, Cleveland, Ohio M.G. Fakih, Duarte, Calif. J.J. Grau, Barcelona H. Gronemeyer, Illkirch D.F. Hayes, Ann Arbor, Mich. C.S. Johnson, Buffalo, N.Y. M.J. Kelley, Durham, N.C. L. Kumar, New Delhi P.J. Loehrer, Indianapolis, Ind. J.R. Marshall, Buffalo, N.Y. S. Monfardini, Milan R. Nagler, Haifa R. Ohno, Nagoya B. Pestalozzi, Zurich H.M. Pinedo, Amsterdam E.A. Repasky, Buffalo, N.Y. A. Semczuk, Lublin E.F. Smit, Amsterdam C.N. Sternberg, Rome R. Stupp, Zurich M.S. Tallman, Chicago, Ill. S. Tanaka, Hiroshima M. Tian, Houston, Tex. D.L. Trump, Buffalo, N.Y. T. Wiegel, Ulm W. Yasui, Hiroshima H. Zhang, Hangzhou City Editor-in-Chief