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Background & Aims The Notch receptor family regulates cell fate through cell-cell communication. CSL ( C BF-1/RBP-jκ, S u(H), L ag-1) drives canonical Notch-mediated gene transcription during cell lineage specification, differentiation, and proliferation in the hematopoietic system, the intestine, the pancreas, and the skin. However, the functional roles of Notch in esophageal squamous epithelial biology are unknown. Methods Normal esophageal keratinocytes were stimulated with calcium chloride to induce terminal differentiation. The squamous epithelia were reconstituted in organotypic 3-dimensional culture, a form of human tissue engineering. Notch was inhibited in culture with a γ-secretase inhibitor or dominant negative mastermind-like 1 (DNMAML1). The roles of Notch receptors were evaluated by in vitro gain-of-function and loss-of-function experiments. Additionally, DNMAML1 was targeted to the mouse esophagus by cytokeratin K14 promoter-driven Cre ( K14Cre ) recombination of Lox - STOP - Lox-DNMAML1 . Notch-regulated gene expression was determined by reporter transfection, chromatin immunoprecipitation assays, quantitative reverse-transcription polymerase chain reaction, Western blotting, immunofluorescence, and immunohistochemistry. Results NOTCH1 (N1) was activated at the onset of squamous differentiation in the esophagus. Intracellular domain of N1 (ICN1) directly activated NOTCH3 (N3) transcription, inducing HES5 and early differentiation markers such as involucrin (IVL) and cytokeratin CK13 in a CSL-dependent fashion. N3 enhanced ICN1 activity and was required for squamous differentiation. Loss of Notch signaling in K14Cre;DNMAML1 mice perturbed esophageal squamous differentiation and resulted in N3 loss and basal cell hyperplasia. Conclusions Notch signaling is important for esophageal epithelial homeostasis. In particular, the cross talk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology.

Inflammatory bowel disease, including ulcerative colitis and Crohn’s disease, substantially increases the risk of colorectal cancer. However, mechanisms linking mucosal inflammation to the sequence of dysplasia are incompletely understood. While studies have demonstrated oxidative damage to the colon, this study tests whether genotoxicity is elicited systemically by acute and chronic intestinal inflammation. In this study, genotoxic endpoints were assessed in peripheral leukocytes (DNA single and double strand breaks and oxidative DNA damage) and normochromatic erythrocytes (micronuclei) during chemical or immune-mediated colitis. During three consecutive cycles of intestinal inflammation induced by dextran sulfate sodium (DSS) administration, genotoxicity to peripheral leukocytes and erythroblasts was detected in both acute and chronic phases of DSS-induced inflammation. Reactive oxygen species mediated oxidative stress and DNA damage was confirmed with positive 8-oxoguanine and nitrotyrosine staining in peripheral leukocytes. Levels of DNA damage generally decreased during remission and increased during treatment, correlating with clinical symptoms and systemic inflammatory cytokine levels. In Gαi2−/− and IL-10−/− transgenic mice susceptible to immune-mediated colitis and inflammation-associated adenocarcinoma, similar levels of peripheral leukocyte and erythroblast genotoxicity were also observed. Moreover, this systemic genotoxicity was observed in mice with subclinical inflammation, which was further elevated in those with severe mucosal inflammation. We propose that mucosal inflammation, by eliciting substantial and ongoing systemic DNA damage, contributes early on to genetic instability necessary for progression to IBD-associated dysplasia and the development of cancer.

Introduction: There exists a highly tumorigenic subset of hepatocellular carcinoma (HCC) cells defined by high expression of CD44 and CD133, and that subset has been reported to contain cancer stem-like cells (CSCs) in HCC. Kruppel-like factor 5 (KLF5) is a transcription factor containing 3 zinc finger domains and one transactivation domain. KLF5 regulates many factors related to cell cycle, migration, inflammation, angiogenesis and stemness and has cancer promoting effects in some cancers. Furthermore, some reports have indicated that KLF5 might have important roles in regulation of CSCs. However, the function of KLF5 in HCC remains to be elucidated. Functional roles of KLF5 in regulation of CSCs in HCC were examined in the present study. Methods: HCC and hepatoblastoma cell lines, Huh7, Alexander and HepG2 cells were analyzed. Anti-CD44 and anti-CD133 antibodies were used to detect CSCs in HCC by fluorescence-activated cell sorting (FACS). Indicated cell population was sorted by FACS Aria III (Becton Dickinson). MTS assay was carried out to determine sensitivity to chemotherapeutic reagents, 5FU and CDDP. RNA sequencing was performed by next generation sequencer, IonTM PGM (Lifetechnologies). Retroviral-mediated gene transfer was used to make a stable cell line overexpressing KLF5. Two independent sequences of siRNA against KLF5 were used to knock-down KLF5. Tumorigenicity was determined by soft-agar colony formation assay and xenograft experiments using athymic nude mice. Results: FACS showed heterogeneous cell population in several HCC cell lines. Sorted CD44High/CD133High cells were significantly more resistant to anti-cancer drugs, 5FU and CDDP, and had more colony forming capacity than CD44Low/CD133Low cells as reported previously. RNA sequencing revealed completely different gene expression profiles between CD44High/CD133High and CD44Low/CD133Low cells, and identified over 500 mRNAs, including KLF5, significantly up-regulated in the sorted CD44High/CD133High cells. Overexpression of KLF5 increased the ratio of CD44High/CD133High cells, and consistent with the upregulation of CD44High/CD133High cells, the KLF5 overexpressing cells were more resistant to the anti-cancer drugs and had more colony forming capacity. By contrast, knock-down of KLF5 by siRNA diminished CD44High/CD133High cell population. Finally, KLF5 overexpressing cells formed tumors more frequently in nude mice. Conclusion: Those data provide a novel mechanistic insight that KLF5 might promote tumorigenicity of HCC by upregulation of CSCs and could be a therapeutic target against CSCs in HCC.

Background: A surplus of reactive oxygen species (ROS) in tissues leads to oxidative stress, resulting in various injurious consequences although ROS plays an important role in normal cellular physiology. As a member of NADPH oxidase family, Duox2 is a major sources of H2O2 for gastrointestinal epithelium. Duox2 expressed in mucosal surfaces is believed as an integral part of host defense system. Duox2 expression was elevated in inflammatory conditions of gastrointestinal and respiratory tracts and human colonic cancer caco-2 cell line. However, the expressions of Duox2 in gastrointestinal cancers are still uncertain. Here we detected the expressions of Duox2 mRNA and protein in the tissues of gastric and colorectal tumors to explore the role of it in the process of gastrointestinal cancers. Methods: The curative tumor tissues from gastric cancer patients (n=30) and colorectal cancer (CRC) patients (n=54) were collected from July 2012 to July 2013 at the first affiliated hospital of Henan university of science and technology. Duox2 protein expressionwas semiquantitatively assessed by immunohistochemical detection in all of both gastric cancer and CRC tissues and their matched normal samples, which were apart away from 5 cm of tumor. Duox2 mRNA expression was evaluated by Real Time PCR (gastric cancer n=16 and CRC n=20). Results: Low level of Duox2 protein expression was found in the epithelium mucosa of gastric and colonic normal tissues, where it was mainly located at the apical membrane in gastric epithelial cells and in the brush border of epithelial cells in colon and rectum. However, the expression of Duox2 protein in both gastric cancer and colorectal cancer were significantly higher than that in each corresponding normal epithelium mucosa (P

Background: A surplus of reactive oxygen species (ROS) in tissues leads to oxidative stress, resulting in various injurious consequences although ROS plays an important role in normal cellular physiology. As a member of NADPH oxidase family, Duox2 is a major sources of H2O2 for gastrointestinal epithelium. Duox2 expressed in mucosal surfaces is believed as an integral part of host defense system. Duox2 expression was elevated in inflammatory conditions of gastrointestinal and respiratory tracts and human colonic cancer caco-2 cell line. However, the expressions of Duox2 in gastrointestinal cancers are still uncertain. Here we detected the expressions of Duox2 mRNA and protein in the tissues of gastric and colorectal tumors to explore the role of it in the process of gastrointestinal cancers. Methods: The curative tumor tissues from gastric cancer patients (n=30) and colorectal cancer (CRC) patients (n=54) were collected from July 2012 to July 2013 at the first affiliated hospital of Henan university of science and technology. Duox2 protein expressionwas semiquantitatively assessed by immunohistochemical detection in all of both gastric cancer and CRC tissues and their matched normal samples, which were apart away from 5 cm of tumor. Duox2 mRNA expression was evaluated by Real Time PCR (gastric cancer n=16 and CRC n=20). Results: Low level of Duox2 protein expression was found in the epithelium mucosa of gastric and colonic normal tissues, where it was mainly located at the apical membrane in gastric epithelial cells and in the brush border of epithelial cells in colon and rectum. However, the expression of Duox2 protein in both gastric cancer and colorectal cancer were significantly higher than that in each corresponding normal epithelium mucosa (P

Background Solid-pseudopapillary neoplasm (SPN), a rare neoplasm of the pancreas, frequently harbors mutations in exon 3 of the cadherin-associated protein beta 1 (CTNNB1) gene. Here, we analyzed SPN tissue for CTNNB1 mutations by deep sequencing using next-generation sequencing (NGS).

Introduction:Notch1-mediated signaling influences cell fate decisions, cell cycle and carcinogenesis via a transcription activation complex consisting of ICN1 (the activated form of Notch1), the co-activatorMAML and the transcription factor CSL. However, the consequences of Notch activation are complex and context dependent. The role of Notch signaling in esophageal epithelial biology andmalignant transformation remain elusive. Methods:Human esophageal keratinocytes immortalized by telomerase (EPC1-hTERT and EPC2-hTERT) as well as non-transformed and transformed derivatives including EPC2T overexpressing EGFR, cyclin D1 and p53R175H; transformed human esophageal keratinocytes carrying a human papilloma virus (HPV)-E6E7 fusion oncogene (EN60) and esophageal squamous cell carcinoma (ESCC) cell lines were genetically engineered to express tetracycline-inducible ICN1; dominant-negative MAML1 (DNMAML1), a genetic pan-Notch inhibitor; or short hairpin RNA (shRNA) directed against CSL, HPV-E7 or cyclin-dependent kinase inhibitor p16INK4A. Notch activity was determined by transfection assays of an 8xCSL-luciferase reporter construct. Notch target genes were determined by quantitative RT-PCR. Western blotting determined retinoblastoma (RB) protein phosphorylation status. Cell proliferation was determined by colorimetric assays. Anchorage-independent colony formation was evaluated in soft agar. Tumor formation was assessed by xenograft transplantation in immunodeficient mice. Senescence associated β-galactosidase (SABG) activity was determined. Results: ICN1 activated CSL-dependent transcription in all cell lines tested and this activation was antagonized by either DNMAML1 expression or CSL knockdown. In response to ICN1 induction, nontransformed cells underwent growth arrest displaying flat and enlarged cell morphology with SABG positive staining (40-80%), suggesting senescence. ICN1-mediated senescence was found to be CSL-dependent and was associated with decreased RB protein phosphorylation and p16INK4A upregulation. ICN1-mediated senescence was inhibited by p16INK4A knockdown, but not the presence of p53R175H. ICN1 failed to induce senescence in EN60 cells expressing EPV-E7 protein or ESCC cell lines lacking the INK4A locus. ICN1 stimulated colony formation and tumor growth in these transformed cell lines. HPV E7 knockdown in EN60 cells resulted in senescence with activation of endogenous Notch1. Conclusions: These novel data indicate that loss of the RB protein-dependent senescence checkpoint function may be permissive for Notch1 to facilitate malignant transformation in human esophageal epithelial cells.

Background: Esophageal adenocarcinoma (EA) is often considered to arise from a clonal stem like population of cells, potentially responsible for its poor prognosis. TGF-β and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. We have demonstrated loss of TGF-β signaling components and activation of Notch signaling in Barrett's esophageal adenocarcinoma; however, their contributions to EA and the mechanisms of their action remain unclear. Methods: Immunoblotting and immunofluorescence were used to evaluate protein expression and localization. Notch targets Hes-1, SOX9 and C-MYC transcription were assayed using their luciferase reporters and Q-PCR. Functional studies were performed in β2SP wild type and mutant MEFs cells and in EA cell lines genetically engineered to express high or low levels of β2SP. Results: Increased levels of Notch signaling Hes1 and Jagged1 occurred in EA tissues and cell lines, compared to normal tissues. Loss of β2SP in MEFs cells increased Hes1 expression by 40 fold. In addition, SOX9, a target gene of Notch signaling and a documented stem cell marker was highly up-regulated in EA tumor cells and tissues. Expression of SOX9 was increased in the absence of β2SP in MEFs cells by 9 fold. Down-regulation of β2SP in SKTG-4, FLO-1 and BE3 EA cells by lentivirus shRNA led to increased SOX9 expression and enhanced nuclear localization of both active intracellular Notch1 domain (ICN1) and SOX9. Reintroducing β2SP into EA cells with knock down β2SP decreased SOX9 promoter activity by 10 fold. Concomitantly, an increased proportion of stem cells in β2SP knock down cells were identified using stem cell marker OCT3/4, indicating expansion of putative cancer stem cells in the absence of β2SP. Most interestingly, we observed a direct interaction between Smad3 and ICN1 via Smad3 MH1 domain by GST-pull down assays and that loss of β2SP increases the binding of Smad3 with ICN1 and induces Notch targets SOX9 and C-MYC transcription and decreased expression of its own targets P21, P27 and E-Cadherin. Conclusions: Our findings suggests that loss of TGF-β signaling adaptor β2SP may switch Smad3 function from tumor suppression to tumor promotion by binding intracellular Notch1 domain (ICN1) and activating Notch signaling. Thereby, a potential therapeutic value for targeted therapy in EA is in the setting of loss of β2SP/TGF-β with active Notch signaling.

Introduction: Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated epithelial-to-mesenchymal transition (EMT) and cancer stem cell functions. We have demonstrated recently that ZEBs are upregulated in esophageal squamous cell carcinoma (ESCC) cells with mesenchymal characteristics and increased malignant potential such as anchorage independent growth, invasiveness and tumorigenicity upon xenograft transplantation (Cancer Res. 2011;71:683647). However, the functional roles of ZEBs in chemotherapeutic resistance remain to be elucidated. Methods: Primary tumor tissues from patients who underwent surgery with or without neoadjuvant chemotherapy were analyzed. ESCC cell lines and In Vitro transformed human esophageal cells EPC2T were treated with either 5-fluorouracil or cisplatin, and subjected to WST-1 colorimetric cell proliferation assays, phase contrast imaging, flow cytometry, quantitative RT-PCR andWestern blotting analyses. Short hairpin RNA was stably transduced by lentivirus to knockdown ZEB1 and ZEB2 in a regulatable manner (Tet-On system). Results: 70% of primary tumor tissues from patients who received neoadjuvant chemotherapy (n=10) contained ZEB1 positive ESCC cells, reminiscent of EMT. By contrast, ZEB1 positive ESCC cells were found in 33% of patients without neoadjuvant chemotherapy (n=51). EMT was present in a subset of cultured ESCC cells. The percentage of mesenchymal cells correlated with downregulation of E-cadherin and upregulation of N-cadherin, Vimentin and ZEBs. Moreover, flow cytometry revealed that the mesenchymal ESCC cells express a high level of CD44, a cancer stem cell marker, implicated in invasion, metastasis and chemoresistance. Interestingly, chemotherapeutic drug sensitivity was reduced as a function of the frequency of mesenchymal subset of the cells when four different cell lines were compared. In two cell lines (TE1 and TE5) with moderate levels of mesenchymal cells, the cells surviving 5-fluorouracil treatment were predominantly mesenchymal. In HCE7 cells displaying fully mesenchymal characteristics, knockdown of either ZEB1 or ZEB2 increased 5-fluorouracil sensitivity. Moreover, TGF-β induced EMT in EPC2T cells, resulted in a significantly increased cell viability rate upon cisplatin treatment and that was antagonized by knockdown of either ZEB1 or ZEB2. Conclusions: These data demonstrate that ZEB1 and ZEB2 confer chemotherapy resistance through EMT-mediated enrichment of a unique subset of ESCC cells defined by a CD44 upregulation. Neoadjuvant therapy may induce ZEB1 positive cells, thus having broader implications upon EMT and cancer therapy.

Background: Esophageal adenocarcinoma (EA) is often considered to arise from a clonal stem like population of cells, potentially responsible for its poor prognosis. TGF-β and Notch signaling pathways play important roles in regulating self-renewal of stem cells and gastrointestinal carcinogenesis. We have demonstrated loss of TGF-β signaling components and activation of Notch signaling in Barrett's esophageal adenocarcinoma; however, their contributions to EA and the mechanisms of their action remain unclear. Methods: Immunoblotting and immunofluorescence were used to evaluate protein expression and localization. Notch targets Hes-1, SOX9 and C-MYC transcription were assayed using their luciferase reporters and Q-PCR. Functional studies were performed in β2SP wild type and mutant MEFs cells and in EA cell lines genetically engineered to express high or low levels of β2SP. Results: Increased levels of Notch signaling Hes1 and Jagged1 occurred in EA tissues and cell lines, compared to normal tissues. Loss of β2SP in MEFs cells increased Hes1 expression by 40 fold. In addition, SOX9, a target gene of Notch signaling and a documented stem cell marker was highly up-regulated in EA tumor cells and tissues. Expression of SOX9 was increased in the absence of β2SP in MEFs cells by 9 fold. Down-regulation of β2SP in SKTG-4, FLO-1 and BE3 EA cells by lentivirus shRNA led to increased SOX9 expression and enhanced nuclear localization of both active intracellular Notch1 domain (ICN1) and SOX9. Reintroducing β2SP into EA cells with knock down β2SP decreased SOX9 promoter activity by 10 fold. Concomitantly, an increased proportion of stem cells in β2SP knock down cells were identified using stem cell marker OCT3/4, indicating expansion of putative cancer stem cells in the absence of β2SP. Most interestingly, we observed a direct interaction between Smad3 and ICN1 via Smad3 MH1 domain by GST-pull down assays and that loss of β2SP increases the binding of Smad3 with ICN1 and induces Notch targets SOX9 and C-MYC transcription and decreased expression of its own targets P21, P27 and E-Cadherin. Conclusions: Our findings suggests that loss of TGF-β signaling adaptor β2SP may switch Smad3 function from tumor suppression to tumor promotion by binding intracellular Notch1 domain (ICN1) and activating Notch signaling. Thereby, a potential therapeutic value for targeted therapy in EA is in the setting of loss of β2SP/TGF-β with active Notch signaling.

Preoperative Biliary Drainage Versus Direct Operation for Pancreatic Tumors Causing Obstructive Jaundice Niels Anthony Van Der Gaag, Erik Rauws, Casper H. van Eijck, Marco Bruno, Erwin van der Harst, Josephus J. Gerritsen, J. W. M. Greve, Michael F. Gerhards, Ignace H. de Hingh, Jean H. Klinkenbijl, Chung Y. Nio, Steve M. de Castro, Olivier R. Busch, Thomas M. Van Gulik, Patrick M. Bossuyt, Dirk J. Gouma