Nobukatsu Horita

The acquisition of malignant potential in colon cancer is regulated by the stabilization of Atonal homolog 1 protein

The transcription factor Atonal homolog 1 (Atoh1) plays crucial roles in the differentiation of intestinal epithelium cells. Although we have reported that the Atoh1 protein was degraded in colon cancer by aberrant Wnt signaling, a recent study has indicated that the Atoh1 protein is expressed in mucinous colon cancer (MC) and signet ring cell carcinoma (SRCC). However, the roles of the Atoh1 protein in MC are unknown. To mimic MC, a mutated Atoh1 protein was stably expressed in undifferentiated colon cancer cells. Microarray analysis revealed the acquisition of not only the differentiated cell form, but also malignant potential by Atoh1 protein stabilization. In particular, Atoh1 enhanced Wnt signaling, resulting in the induction of Lgr5 as a representative stem cell marker with the enrichment of cancer stem cells. Moreover, the fluorescent ubiquitination-based cell cycle indicator system with time-lapse live imaging demonstrated cell cycle arrest in the G0/G1 phase by Atoh1 protein stabilization. In conclusion, the Atoh1 protein regulates malignant potential rather than the differentiation phenotype of MC, suggesting the mechanism by which MC and SRCC are more malignant than non-mucinous adenocarcinoma.

Atonal homolog 1 protein stabilized by tumor necrosis factor α induces high malignant potential in colon cancer cell line

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colitis‐associated colorectal cancer (CAC). CAC cells often develop chemoresistance, resulting in a poorer prognosis than that of sporadic colorectal cancer (CRC). The mechanism by which CAC enhances malignant potential remains unknown. We have previously reported that the proteasomal degradation of the transcription factor Atonal homolog 1 (Atoh1) protein results in the non‐mucinous form of CRC. It also remains unknown whether Atoh1 protein is expressed in CAC. Therefore, in the present study, we investigated whether Atoh1 protein stabilizes in CAC. Consequently, the treatment with TNF‐α stabilized Atoh1 protein through the inactivation of GSK‐3β via Akt, resulting in the mucinous form of CRC cell lines. Atoh1 protein also enriched cancer stem cells with upregulated Lgr5 expression and cells in G0/G1 cell cycle phase, resulting in both the chemoresistance to 5‐fluorouracil and oxaliplatin and the promotion of cell migration. Immunofluorescence of the human mucinous CAC specimens showed the accumulation of NF‐κB p65 at nuclei with the expression of Atoh1 in mucinous cancer. In conclusion, the inflammation associated with carcinogenesis may preserve the differentiation system of intestinal epithelial cell (IEC), resulting in the acquisition of both the mucinous phenotype and high malignant potential associated with the enrichment of cancer stem cell.

Long-term Inflammation Transforms Intestinal Epithelial Cells of Colonic Organoids

Background and Aims Patients with ulcerative colitis [UC] are at an increased risk of developing colitis-associated cancer [CAC], suggesting that continuous inflammation in the colon promotes the transformation of colonic epithelial cells. However, the mechanisms underlying cell transformation in UC remain unknown. We therefore aimed to investigate the effect of long-term inflammation on intestinal epithelial cells [IECs] using organoid culture. Methods IECs were isolated from mouse colon, and were cultured according to a method for a three-dimensional [3D] organoid culture. To mimic chronic inflammation, a mixture of cytokines and bacterial components were added to the medium for over a year. Cell signal intensity was assessed by 3D immunofluorescence. Cell transformation was assessed by microarray with gene set enrichment analysis. Results Stimulation with cytokines resulted in a significant induction of target genes for the nuclear factor [NF]-κB pathway in colonic organoids. Following 60 weeks of continuous stimulation, cell differentiation was suppressed. Continuous stimulation also resulted in significant amplification of NF-κB signalling. Amplified NF-κB signalling by long-term stimulation remained in colonic organoids even 11 weeks after the removal of all cytokines. Some genes were specifically upregulated only in colonic organoids after the removal all cytokines following long-term stimulation. Conclusions Colonic organoids stimulated with cytokines for a prolonged period were established as in vitro model to assess long-term epithelial responses to inflammatory cytokines. Chronic inflammation led to sustained NF-κB signalling activation in colonic organoids, resulting in cell transformation that might be related to the carcinogenesis of CAC in UC.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

BACKGROUND AND AIMS The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. METHODS Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. RESULTS We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. CONCLUSIONS The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

Reduced Human α-defensin 6 in Noninflamed Jejunal Tissue of Patients with Crohn’s Disease:

Background:Mucosal barrier dysfunction is considered a critical component of Crohn’s disease (CD) pathogenesis after the identification of susceptibility genes. However, the precise mechanism underlying mucosal barrier dysfunction has not yet been elucidated. We therefore aimed to elucidate the molecular mechanism underlying the expression of human &agr;-defensin 6 (HD6) in patients with CD. Methods:HD6 expression was induced by the transfection of an atonal homolog 1 (Atoh1) transgene and was assessed by reverse transcription polymerase chain reaction. The HD6 promoter region targeted by Atoh1 and &bgr;-catenin was determined by reporter analysis and chromatin immunoprecipitation assay. HD5/HD6/Atoh1/&bgr;-catenin expression in noninflamed jejunal samples collected by balloon endoscopy from 15 patients with CD and 9 non-inflammatory bowel disease patients were assessed by immunofluorescence. Results:Both promoter activity and gene expression of HD6 was significantly upregulated by the Atoh1 transgene in human colonic cancer cell line. We identified a TCF4 binding site and an E-box site, critical for the regulation of HD6 transcriptional activity by directly binding of Atoh1 in the 200-bp HD6 promoter region. The treatment with &bgr;-catenin inhibitor also decreases HD6 promoter activity and gene expression. Moreover, HD6 expression, but not HD5 expression, was found to be decreased in noninflamed jejunal regions from patients with CD. In HD6-negative crypts, nuclear accumulation of &bgr;-catenin was impaired. Conclusions:HD6 expression was found to be regulated by cooperation between Atoh1 and &bgr;-catenin within the HD6 promoter region. Downregulation of HD6 in noninflamed mucosa may contribute to mucosal barrier dysfunction of patients with CD.

Caudal type homeobox 2 expression induced by leukocytapheresis might be associated with mucosal healing in ulcerative colitis: CDX2 by LCAP for mucosal healing in UC

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with an intractable, recurrent course. Although the goal of UC therapy has recently been to target mucosal healing, the molecular mechanism of mucosal healing remains unknown. In this study, we aimed to elucidate the molecular dynamics related to the proliferation and differentiation of intestinal epithelial cells during cytapheresis therapy in a short duration.

CDX2 expression induced by leukocytapheresis might be associated with mucosal healing in ulcerative colitis

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with an intractable, recurrent course. Although the goal of UC therapy has recently been to target mucosal healing, the molecular mechanism of mucosal healing remains unknown. In this study, we aimed to elucidate the molecular dynamics related to the proliferation and differentiation of intestinal epithelial cells during cytapheresis therapy in a short duration.

Mo1833 Human Alpha-Defensin 6 Regulated by the Cooperation of Beta-Catenin and ATOH1, Might Be the Pathogenesis of Crohn's Disease

Recommended therapy of bacterial infection is generally based on in vitro susceptibility of an organism to an infecting organism. Yet drug bioavailability based on solubility, protein binding and other factors along with host resistance and inflammatory-immune response importantly alter response to treatment. The objective of the study was to establish in vivo animal model to study antimicrobial therapy of C. difficile infection. C. difficile isolate with hyper-virulent factors (toxin A/B plus binary toxin and 18 base pair deletion in tcdC gene) was used in the study. The cultured C. difficile was stained with near infrared (NIR) dye. Stained C. difficilewere determined by using fluorescent microscope and confocal microscope. To monitor stained bacterial trafficking in the gut animals were imaged in real-time. The intestines from treated animals were dissected at the end of imaging study. The bacterial signal intensity and migration pattern were recorded and compared. C. difficile toxin secretion in the different segment of GI system was analyzed. Results showed the C. difficile can be stained by NIR dye. Stained bacteria can be imaged by NIR camera system to track the C. difficile traffic in the gut in real-time. Untreated control animal has different bacteria migration pattern than the animal with C. difficile infection treated with ciprofloxacin. TheNIR florescent probe can be used to visualize C. difficile trafficking in the gastrointentinal tract through culturing, staining and imaging. Fig. 1. Confocal images of C. difficile. Viable bacterial stained with NIR dye (A. Red) and dead bacterial determined with Sytox green (B. Green). Stain efficiency was determined by co-localized all cells (C. Green) and NIR (C. Red). Confocal microscope analyzed NIR dye binding at subcellular level (D). Red and green represent NIR and cell nuclei, respectively. Results demonstrate C. difficile can be stained by NIR dye. Most bacteria remain viable after the staining procedure.

Su1731 Stabilization of ATOH1 Protein by TNF-α in Colitic Cancer Might Acquire the Cancer Stemness

ine treated BxPc3 and Hpac cell lines compared to pre-treatment cell lines. CD24+CD44+ cells sorted from the gemcitabine treated cell lines showed higher migration and invasion ability compared to CD24-CD44cells from the same cell lines. Western blot showed the expression of Hes1, pAKT, and beta-catenin was increased in gemcitabine treated cell lines. The overall survival of pancreatic cancer patients with strong expression of Hes1 was shorter than patients with none or weak expression (21.6 vs 11.1 months p=0.036). Treatment with DAPT reversed the increase of Hes1, beta-catenin expression and CD24+CD44+ cells, and also decreased the migration and invasion ability of gemcitabine treated cell lines. CONCLUSION: Notch pathway was involved in the increase and activation of CSC, which might lead to the treatment failure of pancreatic cancer. Notch pathway is a potential treatment target to improve the survival of pancreatic cancer patients.

Su1736 Live Imaging of Single Cell Reveals Single Stem Cell Dynamics in an Organoid Derived From Murine Small Intestine

G A A b st ra ct s differentiation, thus showing that in analogy to the intestine, esophageal stemness is lost. Using RNA micro-arrays, we identified a gene signature of 47 genes that were lost in both individual cell lines upon induction of ER stress. Of these genes, we found 29 genes to be restricted to the basal layer of the mouse esophagus. Out of these 29, nine genes show expression in only a small proportion of the basal cells, potentially marking stem cells. Conclusion: ER stress depletes esophageal precursor cells. Our in vitro screen combined with in situ hybridization identified nine genes that are specifically expressed in a subset of proliferating genes, thereby potentially marking esophageal stem cells.