Schlafen 11 Is a Novel Target for Mucosal Regeneration in Ulcerative Colitis.

BACKGROUND AND AIMS: Ulcerative colitis [UC] is a chronic inflammatory disease of the colon with an intractable course. Although the goal of UC therapy is to achieve mucosal healing, the pathogenesis of mucosal injury caused by chronic inflammation remains unknown. We therefore aim to elucidate molecular mechanisms of mucosal injury by establishing in vitro and in vivo humanised UC-mimicking models. METHODS: An in vitro model using human colon organoids was established by 60 weeks of inflammatory stimulation. The key gene for mucosal injury caused by long-term inflammation was identified by microarray analysis. An in vivo model was established by xenotransplantation of organoids into mouse colonic mucosa. RESULTS: An in vitro model demonstrated that long-term inflammation induced irrecoverable changes in organoids: inflammatory response and apoptosis with oxidative stress and suppression of cell viability. This model also mimicked organoids derived from patients with UC at the gene expression and phenotype levels. Microarray analysis revealed Schlafen11 [SLFN11] was irreversibly induced by long-term inflammation. Consistently, SLFN11 was highly expressed in UC mucosa but absent in normal mucosa. The knockdown of SLFN11 [SLFN11-KD] suppressed apoptosis of intestinal epithelial cells [IECs] induced by inflammation. Moreover, SLFN11-KD improved the take rates of xenotransplantation and induced the regenerative changes of crypts observed in patients with UC in remission. CONCLUSIONS: In vitro and in vivo UC-mimicking models were uniquely established using human colonic organoids. They revealed that SLFN11 is significant for mucosal injury in UC, and demonstrated its potential as a novel target for mucosal regeneration.

Intestinal phenotype is maintained by Atoh1 in the cancer region of intraductal papillary mucinous neoplasm.

Intraductal papillary mucinous neoplasm (IPMN) is a precancerous lesion of pancreatic cancer. Although there are 4 types of IPMN, among which intestinal-type IPMN is likely to progress into invasive cancer known as colloid carcinoma, no information regarding the involvement of the intestinal phenotype in the carcinogenesis of IPMN exists. The present study was conducted to explore how the intestinal differentiation system is maintained during the tumor progression of intestinal-type IPMN using surgical resection specimens. Results showed that Atoh1, a critical transcriptional factor for intestinal differentiation toward the secretory lineages of intestinal epithelial cells, was expressed in an invasive-grade IPMN. To determine the function of Atoh1 in pancreatic cancer, we generated a pancreatic ductal adenocarcinoma (PDAC) cell line overexpressing Atoh1. In a xenograft model, we successfully induced an IPMN phenotype in PDAC cells via Atoh1 induction. Finally, for the first time, we discovered that GPA33 is expressed in intestinal-type IPMN, thereby suggesting a novel target for cancer therapy. In conclusion, the intestinal differentiation system might be maintained during tumor progression of intestinal-type IPMN. Further analysis of the function of Atoh1 in IPMN might be useful for understanding the molecular mechanism underlying the malignant potential during the tumor progression of IPMN.

TP53 Mutation by CRISPR System Enhances the Malignant Potential of Colon Cancer.

Tumor protein p53 (TP53) mutation is a well-known occurrence at the late phase of carcinogenesis during the adenoma-carcinoma sequence of a sporadic colon cancer. Although numerous reports about clinical information of the patients with colon cancer have suggested that TP53 mutation might be related to various types of malignant potential, the direct effects of this mutation on the malignant potential of colon cancer remain unknown. Notably, no previous report has described a relationship between TP53 mutation and cancer stemness. We therefore aimed to assess the function of a TP53 mutant induced by the CRISPR-Cas9 system in colon cancer cells. In this study, two TP53 mutations, corresponding to exon 3 (TP53E3) and 10 (TP53E10), were generated in LS174T cells derived from a wild-type TP53 human colon cancer via a lentiviral CRISPR-Cas9 system. The loss of function of TP53 resulting from both mutations manifested as resistance to Nutlin3a-induced apoptosis and the downregulation of target genes of TP53. TP53 mutants exhibited an enhanced malignant potential, characterized by accelerated cell growth, invasiveness, chemoresistance, and cancer stemness. Interestingly, TP53E10 but not TP53E3 cells exhibited aberrant transcriptional activity of regenerating family member 1-α (REG1A) and expression of REG1A, resulting in the acquisition of enhanced malignant potential. In conclusion, we demonstrated for the first time that TP53 genomic mutation into human colon cancer cells affects the malignant potential. IMPLICATIONS: These findings suggest that both a loss of function and an aberrant gain of function of TP53 might promote high malignant potentials at the late phase of carcinogenesis in colon cancer.

Establishment of a system to evaluate the therapeutic effect and the dynamics of an investigational drug on ulcerative colitis using human colonic organoids.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with an intractable, recurrent course. The goal of UC therapy is to target mucosal healing because immune-suppressive therapy for UC frequently results in relapse. However, few drugs directly target mucosal healing. We, therefore, aim to evaluate the therapeutic effect of an investigational drug on intestinal epithelial cells in an in vitro UC model using human colonic organoids. METHODS: Colonic organoids were isolated from human colon and cultured. A mixture of cytokines and bacterial components were used to mimic UC in humans. The effect of the investigational drug on colonic organoid was evaluated by microarray analysis and 3D immunofluorescence. The enrichment of stem cells was assessed with a colony formation assay. RESULTS: Inflammatory stimulation resulted in a significant induction of inflammatory-related genes in colonic organoids whereas cell differentiation was suppressed. Treatment with the investigational drug KAG-308 showed reciprocal dynamics of gene expression to inflammatory stimulation, which resulted in not only the suppression of immune response but also the promotion of cellular differentiation towards secretory lineages. Moreover, SPDEF and Reg4 were identified as novel targets for the enrichment of intestinal epithelial stem cells and mucosal healing. CONCLUSIONS: The establishment of in vitro UC model using human colonic organoid could reveal the effects and targets of investigational drugs in intestinal epithelial cells under inflammation conditions. Further maturation of this system might be more efficient to predict the effect on UC, as compared with the use of animal model, for the development of new drugs.

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.

Efficacy of short period, low dose oral prednisolone for the prevention of stricture after circumferential endoscopic submucosal dissection (ESD) for esophageal cancer.

BACKGROUND AND AIMS: Endoscopic submucosal dissection (ESD) was developed in Japan and has been performed on many patients with early stage esophageal cancer; however quality of life in patients with postoperative stricture is drastically decreased and repeat, periodic endoscopic balloon dilatation (EBD) is usually required over long periods. In this study, we evaluate the efficacy of short period, low dose oral prednisolone in controlling post-procedural esophageal stricture. PATIENTS AND METHODS: In total, 33 patients who underwent semicircular or complete circular ESD for esophageal superficial squamous cell carcinoma were included in this study. They were divided into two groups: those who underwent large-circumference ESD with no preventative treatment for stricture (ESD alone group) and those who received systemic steroid treatment for stricture (oral prednisolone group). We compared the two groups in terms of stricture rate and total number of EBD sessions. The ESD alone group underwent no preventative treatment. The oral prednisolone group started with 30 mg/day prednisolone on the second day post-ESD, and continued with a gradually tapering prednisolone dose, finally discontinuing systemic steroid administration 3 weeks later. RESULTS: The stricture rate after ESD was significantly lower in the oral prednisolone group (3 of 17 patients; 17.6 %) than in the ESD alone group (11 of 16 patients; 68.7 %) (P < 0.01). The number of EBD sessions was significantly lower in the oral prednisolone group than in the ESD alone group (median 4.6, range 2 - 10 vs. median 8.1, range 1 - 18; P < 0.01). CONCLUSION: Short period, low dose oral prednisolone showed promising results for the prevention of stricture after ESD for early stage esophageal cancers.