Eri Kameta

Intestine-specific homeobox (ISX) induces intestinal metaplasia and cell proliferation to contribute to gastric carcinogenesis.

BackgroundHelicobacter pylori induces chronic inflammation and intestinal metaplasia (IM) through genetic and epigenetic changes and activation of intracellular signaling pathways that contribute to gastric carcinogenesis. However, the precise mechanism of IM in gastric carcinogenesis has not been fully elucidated. We previously found that intestine-specific homeobox (ISX) mRNA expression increased in organoids cultured from Helicobacter-infected mouse mucosa. In this study, we elucidate the role of ISX in the development of IM and gastric carcinogenesis.MethodsISX expression was assessed in Helicobacter-infected mouse and human gastric mucosa. MKN45 gastric cancer cells were co-cultured with H. pylori to determine whether Helicobacter infection induced ISX expression. We established stable MKN45 transfected cells expressing ISX (Stable-ISX MKN45) and performed a spheroid colony formation assay and a xenograft model. We performed ISX immunohistochemistry in cancer and adjacent gastric tissues.ResultsISX expression was increased in mouse and human gastric mucosa infected with Helicobacter. The presence of IM and H. pylori infection in human stomach was correlated with ISX expression. H. pylori induced ISX mRNA and protein expression. CDX1/2, cyclinD1, and MUC2 were upregulated in Stable-ISX MKN45, whereas MUC5AC was downregulated. Stable-ISXMKN45 cells formed more spheroid colonies, and had high tumorigenic ability. ISX expression in gastric cancer and adjacent mucosa were correlated.ConclusionsISX expression induced by H. pylori infection may lead to IM and hyperproliferation of gastric mucosa through CDX1/2 and cyclinD1 expression, contributing to gastric carcinogenesis.

Efficacy of plastic stent placement inside bile ducts for the treatment of unresectable malignant hilar obstruction (with videos)

Recent reports have addressed the utility of plastic stent (PS) placement inside bile ducts for treating biliary obstructions. Here, we evaluated the utility and safety of PS placement inside bile ducts for treating unresectable malignant hilar biliary obstruction.

Helicobacter-induced gastric inflammation alters the properties of gastric tissue stem/progenitor cells

BackgroundAlthough Helicobacter-induced gastric inflammation is the major predisposing factor for gastric carcinogenesis, the precise mechanism by which chronic gastritis causes gastric cancer remains unclear. Intestinal and spasmolytic polypeptide-expressing metaplasia (SPEM) is considered as precancerous lesions, changes in epithelial tissue stem/progenitor cells after chronic inflammation has not been clarified yet. In this study, we utilized three-dimensional gastric epithelial cell culture systems that could form organoids, mimicking gastric epithelial layer, and characterized the changes in epithelial cells after chronic Helicobacter felis infection.MethodsWe used three mice model; 1) long-term H. felis infection, 2) H. felis eradication, and 3) MNU chemical carcinogenesis model. We performed cRNA microarray analysis after organoid culture, and analyzed the effects of chronic gastric inflammation on tissue stem cells, by the size of organoid, mRNA expression profile and immunohistochemical analysis.ResultsThe number of organoids cultured from gastric epithelial cells was significantly higher in organoids isolated from H. felis-infected mice compared with those from uninfected gastric mucosa. Based on the mRNA expression profile, we found that possible stem cell markers such as Cd44, Dclk1, and genes associated with the intestinal phenotype, such as Villin, were increased in organoids isolated from H. felis-infected mucosa compared with the control. The upregulation of these genes were cancelled after H. felis eradication. In a xenograft model, tumors were generated only from organoids cultured from carcinogen-treated gastric mucosa, not from H. felis infected mucosa or control organoids.ConclusionsOur results suggested that, as a possible mechanism of gastric carcinogenesis, chronic inflammation induced by H. felis infection increased the number of tissue stem/progenitor cells and the expression of stem cell markers. These findings suggest that chronic inflammation may alter the direction of differentiation toward undifferentiated state and that drawbacks may enable cells to redifferentiate to intestinal metaplasia or neoplasia.

Diagnosis of pancreatic lesions collected by endoscopic ultrasound-guided fine-needle aspiration using next-generation sequencing

Endoscopic ultrasound-guided fine-needle aspiration (EUF-FNA) has improved the diagnosis of pancreatic lesions. Next-generation sequencing (NGS) facilitates the production of millions of sequences concurrently. Therefore, in the current study, to improve the detectability of oncogenic mutations in pancreatic lesions, an NGS system was used to diagnose EUS-FNA samples. A total of 38 patients with clinically diagnosed EUS-FNA specimens were analyzed; 27 patients had pancreatic ductal adenocarcinoma (PDAC) and 11 had non-PDAC lesions. DNA samples were isolated and sequenced by NGS using an Ion Personal Genome Machine system. The Cancer Hotspot Panel v2, which includes 50 cancer-related genes and 2,790 COSMIC mutations, was used. A >2% mutation frequency was defined as positive. KRAS mutations were detected in 26 of 27 PDAC aspirates (96%) and 0 of 11 non-PDAC lesions (0%). The G12, G13, and Q61 KRAS mutations were found in 25, 0, and 1 of the 27 PDAC samples, respectively. Mutations were confirmed by TaqMan® polymerase chain reaction analysis. TP53 mutations were detected in 12 of 27 PDAC aspirates (44%). SMAD4 was observed in 3 PDAC lesions and cyclin-dependent kinase inhibitor 2A in 4 PDAC lesions. Therefore, the current study was successfully able to develop an NGS assay with high clinical sensitivity for EUS-FNA samples.

Activation of Signal Transduction and Activator of Transcription 3 Signaling Contributes to Helicobacter-Associated Gastric Epithelial Proliferation and Inflammation

Background/Aim Although IL-6-mediated activation of the signal transduction and activator of transcription 3 (STAT3) axis is involved in inflammation and cancer, the role of STAT3 in Helicobacter-associated gastric inflammation and carcinogenesis is unclear. This study investigated the role of STAT3 in gastric inflammation and carcinogenesis and examined the molecular mechanism of Helicobacter-induced gastric phenotypes. Methods To evaluate the contribution of STAT3 to gastric inflammation and carcinogenesis, we used wild-type (WT) and gastric epithelial conditional Stat3-knockout (Stat3Δgec) mice. Mice were infected with Helicobacter felis and euthanized at 18 months postinfection. Mouse gastric organoids were treated with recombinant IL-6 (rIL-6) or rIL-11 and a JAK inhibitor (JAKi) to assess the role of IL-6/STAT3 signaling in vitro. Results Inflammation and mucous metaplasia were more severe in WT mice than in Stat3Δgec mice. The epithelial cell proliferation rate and STAT3 activation were increased in WT mice. Application of rIL-6 and rIL-11 induced expression of intestinal metaplasia-associated genes, such as Tff2; this induction was suppressed by JAKi administration. Conclusions Loss of STAT3 signaling in the gastric mucosa leads to decreased epithelial cell proliferation, atrophy, and metaplasia in the setting of Helicobacter infection. Therefore, activation of STAT3 signaling may play a key role in Helicobacter-associated gastric carcinogenesis.

c-Jun N-terminal kinase in pancreatic tumor stroma augments tumor development in mice

Pancreatic ductal adenocarcinoma (PDAC) is a life‐threatening disease and there is an urgent need to develop improved therapeutic approaches. The role of c‐Jun N‐terminal kinase (JNK) in PDAC stroma is not well defined even though dense desmoplastic reactions are characteristic of PDAC histology. We aimed to explore the role of JNK in PDAC stroma in mice. We crossed Ptf1aCre/+;KrasG12D/+ mice with JNK1−/− mice to generate Ptf1aCre/+;KrasG12D/+;JNK1−/− (Kras;JNK1−/−) mice. Tumor weight was significantly lower in Kras;JNK1−/− mice than in Kras;JNK1+/− mice, whereas histopathological features were similar. We also transplanted a murine PDAC cell line (mPC) with intact JNK1 s.c. into WT and JNK1−/− mice. Tumor diameters were significantly smaller in JNK1−/− mice. Phosphorylated JNK (p‐JNK) was activated in α‐smooth muscle actin (SMA)‐positive cells in tumor stroma, and mPC‐conditioned medium activated p‐JNK in tumor‐associated fibroblasts (TAF) in vitro. Relative expression of Ccl20 was downregulated in stimulated TAF. Ccl20 is an important chemokine that promotes CD8+ T‐cell infiltration by recruitment of dendritic cells, and the number of CD8+ T cells was decreased in Kras;JNK1+/− mice compared with Kras;JNK1−/− mice. These results suggest that the cancer secretome decreases Ccl20 secretion from TAF by activation of JNK, and downregulation of Ccl20 secretion might be correlated with reduction of infiltrating CD8+ T cells. Therefore, we concluded that inhibition of activated JNK in pancreatic tumor stroma could be a potential therapeutic target to increase Ccl20 secretion from TAF and induce accumulation of CD8+ T cells, which would be expected to enhance antitumor immunity.

Sa1978 Overexpression of HER2 Converted the KRAS-Driven PanIN Lesions to IPMC Phenotype

Background: Enzymes that initiate tryptophan metabolism along the kynurenine pathway (KP) are commonly overexpressed in colorectal cancer (CRC). One of these enzymes, IDO1, has recently been shown to promote tumorigenesis in a mouse model of colitis associated cancer and promote nuclear translocation of β-catenin and proliferation in CRC cells. The current investigation is aimed at identifying the signaling pathways activated by various KP metabolites and determining if a similar effect exists in non-neoplastic primary colonic epithelial cells (CEC). Methods: In vitro models of human CRC using various CRC cell lines (HT29, HCT116 and SW480) as well as human colon primary CECs cultured in growth factor (Wnt3a, Noggin, R-Spondin) enriched media were used. All experiments were performed in serum and growth factor free culture media with or without the addition of KP metabolites such as Kynurenine (Kyn), Quinolinic Acid (QA) and Picolinic Acid (PA). Expression of mRNA and protein as well as their phosphorylation status was quantified at multiple time points, and cell proliferation was measured by MTT assay. Results: Addition of Kyn, QA and PA (100 μM) rapidly induced (5 min) phosphorylation of AKT (Ser473) and LRP (Ser1490), a required co-receptor for activating Wnt/β-catenin signaling. The KP metabolites also rapidly inactivated GSK-3β as indicated by phosphorylation at Ser9 and induced expression of nuclear β-catenin (P-Ser552). These changes were followed by an induction of CyclinD1 expression at 12-36 hours along with an increase in CRC cell proliferation after Kyn and QA application. However, with PA, no functional changes in proliferation were observed. In contrast to CRC cells, KP metabolites treatment of primary human CECs led to comparatively moderate and delayed changes in AKT, GSK-3β and βcatenin phosphorylation. Additionally, no change in LRP phosphorylation or effect on primary CEC proliferation was observed. Conclusions: KP metabolites induce rapid and reversible changes in the activation state of cellular machinery linked to CRC cell proliferation and neoplastic growth. While similarities exist between initial signaling events of KP metabolites, important distinctions were observed in their ultimate effects on proliferation of CRC and primary colonic epithelial cells. Together these observations highlight the role of the KP metabolites in β-catenin signaling and form the basis of future interrogation of this pathway for therapeutic targeting of colorectal cancer that may spare the normal epithelium.