Eri Oda

Involvement of the IRF family transcription factor IRF-3 in virus-induced activation of the IFN-β gene

The virus‐induced activation of interferon α/β (IFN‐α/β) gene transcription is essential for host defense. The IFN‐β promoter is controlled primarily by the virus‐inducible enhancer elements, the IRF‐Es. Here we show that IRF‐3, an IRF family transcription factor, translocates to the nucleus from the cytoplasm upon virus infection in NIH/3T3 cells. The nuclear IRF‐3 is phosphorylated, interacts with the co‐activators CBP/p300, and binds specifically to the IFN‐β IRF‐E. Furthermore, overexpression of IRF‐3 causes a marked increase in virus‐induced IFN‐β mRNA expression. Thus, IRF‐3 is a candidate transcription factor mediating the activation of the IFN‐β gene.

Type I interferons are essential mediators of apoptotic death in virally infected cells

The interferons (IFNs) have been extensively studied in the context of host defence against viral infection. In the established model of IFN action, virally infected cells secrete type I IFNs (IFN‐α/β) which induce an antiviral state in uninfected cells. However, it is not clear how IFNs function on the infected cells. It has been reported that cells infected by some viruses die by apoptosis.

Reprimo, a new candidate mediator of the p53-mediated cell cycle arrest at the G2 phase.

A novel gene, Reprimo, in which induction in cells exposed to X-irradiation is dependent on p53 expression, has been isolated. Ectopic p53 expression results in the induction of its mRNA. Reprimo is a highly glycosylated protein and, when ectopically expressed, it is localized in the cytoplasm and induces G2 arrest of the cell cycle. In the arrested cells, both Cdc2 activity and nuclear translocation of cyclin B1 are inhibited, suggesting the involvement of Reprimo in the Cdc2·cyclin B1 regulation pathway. Thus, Reprimo may be a new member involved in the regulation of p53-dependent G2 arrest of the cell cycle.

Functionally inactivating point mutation in the tumor‐suppressor IRF‐1 gene identified in human gastric cancer

Loss of heterozygosity (LOH) observed in human tumors strongly suggests the existence of (a) tumor‐suppressor gene(s) at the concerned locus. A series of studies has revealed that LOH on the long arm of chromosome 5 (5q) frequently occurs in differentiated gastric adenocarcinomas. Furthermore, it has been shown that the interferon regulatory factor‐1 (IRF‐1) locus on chromosome 5q31.1 is one of the common minimal regions of LOH in these cancers. IRF‐1 is a transcriptional activator that shows tumor‐suppressor activity in the mouse. In the present study, we examined the sequence of the IRF‐1 gene in 9 cases of histologically differentiated gastric adenocarcinomas, all of which exhibited LOH at the IRF‐1 locus. We identified a mis‐sense mutation in the residual allele in one case. This mutated form of IRF‐1 showed markedly reduced transcriptional activity. In addition, over‐expression of wild‐type IRF‐1 induced cell‐cycle arrest, whereas such activity was attenuated in the mutant IRF‐1. These results suggest that the loss of functional IRF‐1 is critical for the development of human gastric cancers. Int. J. Cancer 77:522–527, 1998.

Critical role for constitutive type I interferon signaling in the prevention of cellular transformation

Interferons‐α/β, which are produced upon viral infection, are key soluble factors for the establishment of an antiviral state, but are also produced at low levels in the absence of infection. Herein, we demonstrate that a weak signal by these constitutively produced IFN‐α/β show a preventive role in cellular transformation. Ifnar1‐deficient (Ifnar1−/–) MEF, which are devoid of IFN‐α/β signal, undergo a spontaneous transformation during long‐term cell culture. Similar to Irf1−/– MEF, primary Ifnar1−/– MEF become tumorigenic in nude mice by the expression of activated c‐Ha‐Ras oncoprotein. However, Ifnar1−/– MEF do not show any abnormal growth properties. A similar observation is made in Ifnb−/– MEF that fail to produce constitutive IFN‐α/β, whereas such a transforming property is not found in MEF that lack any of the IFN receptor downstream molecules including Stat1, IRF9 and IRF1. Furthermore, Ifnar1−/– mice develop chemically‐induced skin papilloma more severely than wild‐type mice. In addition, the expression levels of IFNAR1 mRNA are significantly decreased in human gastric cancer tissues. These results suggest a cell‐intrinsic role of the weak signal by constitutively produced IFN‐α/β to prevent cells from transformation, which may be mediated by a hitherto‐unknown pathway(s) downstream of the IFN‐α/β receptor. (Cancer Sci 2009; 100: 449–456)

G10BP, an E1A-inducible negative regulator of Sp1, represses transcription of the rat fibronectin gene.

Downregulation of the fibronectin (FN) gene in a rat 3Y1 derivative cell line, XhoC, transformed by the adenovirus E1A and E1B genes seems to be caused by the induction of a negative regulator, G10BP, which binds to three G-rich sequences in the promoter (T. Nakamura, T. Nakajima, S. Tsunoda, S. Nakada, K. Oda, H. Tsurui, and A. Wada, J. Virol. 66:6436-6450, 1992). These are the G10 stretch and two GC boxes consisting of the G10 stretch with one internal C residue insertion. The recognition sequences of G10BP and Sp1 (GGGCGG) overlap in these GC boxes. To analyze the mechanism of the downregulation, G10BP was purified by DNA affinity chromatography, and its molecular mass was estimated to be about 30 kDa. The promoter was modified by substituting the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, and by replacing the Sp1 motif by the T stretch. Transcription of FN promoter-chloramphenicol acetyltransferase fusion genes carrying the base substitution in one or more of these G-rich sequences both in vivo and in vitro revealed that the base substitution in any G-rich sequence results in reduction of promoter activity, although the downstream GC box (GCd) plays a primary role. The addition of G10BP severely inhibited the activities of the FN promoters carrying the wild-type GCd in vitro, while the promoters carrying the mutant GCd were unaffected. The binding affinity of G10BP and Sp1 to each of the G-rich sequences, analyzed by gel shift assays, indicated that G10BP binds strongly to the GCd, moderately to the G10 stretch, and weakly to GCu, while Sp1 binds strongly to GCu, moderately to GCd, and weakly to the G10 stretch. Sp1 binding to GCd and the G10 stretch was inhibited by G10BP, while binding to GCu was unaffected. These results indicate that FN gene transcription is inhibited in XhoC cells primarily by exclusion of Sp1 binding to GCd by G10BP and that G10BP is a new class of Sp1 negative regulator.

INDUCTION OF SP1 IN DIFFERENTIATING HUMAN EMBRYONAL CARCINOMA CELLS TRIGGERS TRANSCRIPTION OF THE FIBRONECTIN GENE

ABSTRACT Cells of the human embryonal carcinoma line NEC14 proliferate as densely packed clusters consisting of small, polygonal stem cells and do not express a detectable level of fibronectin (FN). Upon induction of differentiation by treatment withN,N′-hexamethylene bisacetamide (HMBA), the level of FN mRNA increased steeply within 24 h and FN began to be accumulated, along with the organization of actin filaments in the cells. The FN promoter elements required for the activation were analyzed in reference to a cluster of GC boxes by using the chloramphenicol acetyltransferase (CAT) gene fused to 5′ sequential-deletion derivatives of the promoter and promoters carrying base substitutions in the GC boxes. Among four GC boxes, GC boxes 2 and 3 had the greatest effect on promoter activation, and base substitutions in these GC boxes resulted in 80% reduction in promoter activity. The pattern of DNA-protein complex formation with these GC boxes changed drastically after induction of differentiation. The extract prepared from undifferentiated NEC14 cells formed fast-migrating complexes (UnD complexes), while the extract prepared from NEC14 cells treated with HMBA for 24 h formed slow-migrating complexes containing Sp1. Both complexes were formed predominantly with GC box 2. Base substitutions within the GC boxes completely abolished the formation of both UnD and Sp1 complexes. Consistent with these changes, the Sp1 level increased steeply within 24 h. Induction of Sp1 expression in NEC14 cells effectively stimulated the promoter activity of the transfected FN promoter-CAT constructs. These results indicate that activation of the FN promoter in differentiating NEC14 cells occurs by the steep induction of Sp1, which prevents an undifferentiated cell factor from binding to the Sp1 sites.

Cloning and Characterization of a GC-Box Binding Protein, G10BP-1, Responsible for Repression of the Rat Fibronectin Gene

ABSTRACT Fibronectin (FN) is an extracellular matrix protein that connects the extracellular matrix to intracellular cortical actin filaments through binding to its cell surface receptor, α5β1, a member of the integrin superfamily. The expression level of FN is reduced in most tumor cells, facilitating their anchorage-independent growth by still unclarified mechanisms. The cDNA clone encoding G-rich sequence binding protein G10BP-1, which is responsible for repression of the rat FN gene, was isolated by using a yeast one-hybrid screen with the G10 stretch inserted upstream of the HIS3 and lacZgene minimal promoters. G10BP-1 comprises 385 amino acids and contains two basic regions and a putative zipper structure. It has the same specificity of binding to three G-rich sequences in the FN promoter and the same size as the G10BP previously identified in adenovirus E1A- and E1B-transformed rat cells. Expression of G10BP-1 is cell cycle regulated; the level was almost undetectable in quiescent rat 3Y1 cells but increased steeply after growth stimulation by serum, reaching a maximum in late G1. Expression of FN mRNA is inversely correlated with G10BP-1 expression, and the level decreased steeply during G1-to-S progression. This down regulation was strictly dependent on the downstream GC box (GCd), and base substitutions within GCd abolished the sensitivity of the promoter to G10BP-1. In contrast, the level of Sp1, which competes with G10BP for binding to the G-rich sequences, was constant throughout the cell cycle, suggesting that the concentration of G10BP-1 relative to that of Sp1 determines the expression level of the FN gene. Preparation of glutathione S-transferase pulldowns of native proteins from the cell extracts containing exogenously or endogenously expressed G10BP-1, followed by Western blot analysis, showed that G10BP-1 forms homodimers through its basic-zipper structure.

Significance of lymphadenectomy with splenectomy in radical surgery for advanced (pT3/pT4) remnant gastric cancer

BACKGROUND To date, the optimal surgical strategy for remnant gastric cancer has not been determined. The purpose of this study was to clarify the significance of lymphadenectomy with splenectomy in remnant gastric cancer surgery. METHODS This retrospective cohort study was conducted at the Kumamoto Regional Medical Center. The primary endpoint was overall survival after surgery. We retrospectively analyzed the clinicopathologic features, surgical treatments, and long-term prognosis of remnant gastric cancer patients treated with total gastrectomy. RESULTS A total of 80 patients with gastric cancer in the remnant stomach after distal gastrectomy and who underwent total gastrectomy were enrolled in the study. Splenectomy was performed in 38 patients. Lymph node metastasis in the splenic hilum was not observed in the patients with pT1/pT2 tumors, whereas nodal metastasis at the splenic hilum was detected in 30.4% of the patients with pT3/pT4 tumors. The survival rate of the patients with pT3/pT4 tumors who underwent splenectomy was significantly higher than that of the patients who did not undergo splenectomy, although there was no difference in the patients with pT1/pT2 tumors. Among the patients classified as R0, the survival rate of the patients with pT3/pT4 tumors who underwent splenectomy was significantly higher than that of the patients who did not undergo splenectomy. CONCLUSION Lymphadenectomy with splenectomy in radical surgery is beneficial for patients with advanced (pT3/pT4) remnant gastric cancer.

Abstract 1336: Significance of SERPINE2 expression in peritoneal metastasis in gastric carcinoma

Background: Peritoneal metastasis is one of the most frequent causes of death in patients with advanced gastric carcinoma (GC). However, molecular mechanisms driving peritoneal dissemination still remain poorly understood. Here, we aimed to give new insights to the molecular mechanisms that drive the peritoneal dissemination of GC. This study identified candidates of potential regulators of peritoneal dissemination and focused on the significance of one of them in vitro experiments and clinical samples. Methods: We utilized previously reported combined expression analysis with two cell lines and samples and 200 GC patients to identify driver genes of peritoneal dissemination. The cell lines consist of HSC58, an established cell lines from scirrhous type primary GC and its progeny cell line, 58As9, with in vivo-selected highly peritoneal metastatic potential. In vitro experiments, we utilized the two gastric cancer cell lines, 58As9 and NUGC4 and evaluated the effect of SERPINE2 knockdown on proliferation, spheroid formation, invasion, and motility. As for evaluation of clinical samples, 176 primary gastric carcinomas were included, which were obtained from patients who underwent curative gastrectomy in Kumamoto university between 2005 and 2015. We evaluated expression of SERPINE2 in immunohistochemistry and divided the cohort into two groups by SERPINE2 expression and analyzed relationship with clinicopathological factors and survival. Results: Enrichment analyses of the profile of the peritoneal dissemination-associated expression signature revealed that poor prognosis and peritoneal metastasis was related with the two pathways, focal adhesion (FDR q=0.00195) and extracellular matrix (FDR q=0.00195). Among the genes related with extracellular matrix, Serine Proteinase Inhibitor, Clade E, Member 2 (SERPINE2) was found to be highly expressed in 58As9 compared with HSC58, and we focused on SERPINE2 as a candidate of potential regulators of peritoneal dissemination. In vitro experiments, the SERPINE2 knockdown was related with decrease in cell motility and invasion, but not with decrease in proliferation, spheroid formation. Immunohistochemistry analysis of the clinical samples revealed higher SERPINE2 expression was significantly related with invasion of serosa and lymphatic vessels, and lymph node matastasis, and peritoneal metastatic metastasis. Survival analysis also revealed SERPINE2 expression was related with poor peritoneal recurrence-free survival (p=0.038), and peritoneal metastasis-specific survival (p=0.036). Conclusion: We found SERPINE2 as a candidate of driving molecules of peritoneal metastasis partly because it promotes invasive potential. Further study including in vivo study is required to reveal relationship of SERPINE2 with the other molecules and pathways. Citation Format: Daisuke Kuroda, Junji Kurashige, Hiroshi Sawayama, Masaaki Iwatsuki, Tomoyuki Uchihara, Tasuku Toihata, Eri Oda, Taisuke Yagi, Tsugio Eto, Keisuke Miyake, Mayuko Ouchi, Kenichi Nakamura, Koichi Kinoshita, Kousuke Mima, Takatsugu Ishimoto, Yasuo Sakamoto, Yoshifumi Baba, Naoya Yoshida, Koshi Mimori, Hideo Baba. Significance of SERPINE2 expression in peritoneal metastasis in gastric carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1336. doi:10.1158/1538-7445.AM2017-1336