Yoshihito Kano

Suppression of hath1 gene expression directly regulated by hes1 via notch signaling is associated with goblet cell depletion in ulcerative colitis

Background: The transcription factor Atoh1/Hath1 plays crucial roles in the differentiation program of human intestinal epithelium cells (IECs). Although previous studies have indicated that the Notch signal suppresses the differentiation program of IEC, the mechanism by which it does so remains unknown. This study shows that the undifferentiated state is maintained by the suppression of the Hath1 gene in human intestine. Methods: To assess the effect of Notch signaling, doxycycline‐induced expression of Notch intracellular domain (NICD) and Hes1 cells were generated in LS174T. Hath1 gene expression was analyzed by quantitative reverse‐transcription polymerase chain reaction (RT‐PCR). Hath1 promoter region targeted by HES1 was determined by both reporter analysis and ChIP assay. Expression of Hath1 protein in ulcerative colitis (UC) was examined by immunohistochemistry. Results: Hath1 mRNA expression was increased by Notch signal inhibition. However, Hath1 expression was suppressed by ectopic HES1 expression alone even under Notch signal inhibition. Suppression of the Hath1 gene by Hes1, which binds to the 5′ promoter region of Hath1, resulted in suppression of the phenotypic gene expression for goblet cells. In UC, the cooperation of aberrant expression of HES1 and the disappearance of caudal type homeobox 2 (CDX2) caused Hath1 suppression, resulting in goblet cell depletion. Conclusions: The present study suggests that Hes1 is essential for Hath1 gene suppression via Notch signaling. Moreover, the suppression of Hath1 is associated with goblet cell depletion in UC. Understanding the regulation of goblet cell depletion may lead to the development of new therapy for UC. (Inflamm Bowel Dis 2011;)

Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells.

Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.

Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation

Significance Despite the well-established connection between Ras and Src, there currently is no evidence of direct interaction between these two proteins. We show here that Src binds to and phosphorylates GTP-loaded Ras on a conserved Y32 residue within the switch I region. It has been shown that Raf binds to Ras with an affinity 1,000-fold greater than that of GAP. However, it has remained unclear how GAP is able to outcompete Raf for Ras upon Raf displacement. We show here that Y32 phosphorylation inhibits Raf binding to Ras and concomitantly promotes GAP association and GTP hydrolysis, thereby ensuring unidirectionality to the Ras GTPase cycle. These findings reveal new fundamental mechanistic insight into how Src negatively regulates Ras. Mutations in Ras GTPase and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers and also have been identified in several familial developmental syndromes. Over the past few decades it has become clear that the activity or the oncogenic potential of Ras is dependent on the nonreceptor tyrosine kinase Src to promote the Ras/Raf/MAPK pathway essential for proliferation, differentiation, and survival of eukaryotic cells. However, no direct relationship between Ras and Src has been established. We show here that Src binds to and phosphorylates GTP-, but not GDP-, loaded Ras on a conserved Y32 residue within the switch I region in vitro and that in vivo, Ras-Y32 phosphorylation markedly reduces the binding to effector Raf and concomitantly increases binding to GTPase-activating proteins and the rate of GTP hydrolysis. These results suggest that, in the context of predetermined crystallographic structures, Ras-Y32 serves as an Src-dependent keystone regulatory residue that modulates Ras GTPase activity and ensures unidirectionality to the Ras GTPase cycle.

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.

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.

Abstract B13: Novel treatment strategy for pancreatic cancer by targeting the ‘undruggable’ Ras oncoprotein

Background and Aims: Mutations in RAS and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers, including up to 25% of lung cancers and over 90% of pancreatic cancers. Ras function as ‘molecular switches’ in a number of signaling pathways that regulate vital cellular functions. Over the past few decades, it has become clear that the activity or the oncogenic potential of Ras is dependent on the non-receptor tyrosine kinase Src to regulate essential cellular pathways for proliferation, differentiation and survival of eukaryotic cells. Recently, we showed that Src binds to and phosphorylates Ras on a conserved tyrosine residue at position 32 within the switch I region to promote Ras GTPase activity. We then identified Shp2 as the critical tyrosine phosphatase that is responsible for the dephosphorylation and activation of Ras. However, it is unknown whether pharmacologic manipulation of Shp2 activity would suppress the growth of Ras-driven pancreatic cancer (PDACs), a highly aggressive disease currently without effective treatment options or cure. Methods: We generated the available Ras, Src, and SHP2 reagents (wild-type, oncogenic, truncation, dominant-negative, catalytically active or inactive, mutants) to determine the structure/function relationship between Ras, Src, and SHP2. The interaction was assessed by immunoprecipitation and western blotting. We utilized several PDAC cell lines and patient-derived xenografts cell lines (PDX-C). The cell growth was evaluated by Alarmar Blue assay. Results: We identified SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signaling. In comparison to K-Ras WT pancreatic cancer cells, the level of phospho-Shp2 was observed to be higher in K-Ras mutated cell lines in both absence and presence of growth factors. Pharmacologic inhibition of Shp2 using a specific cell-permeable Shp2 inhibitor decreased the level of phosphorylation of Shp2 as well as downstream signaling such as pAKT and pERK especially in the presence of growth factor. Notably, we show that treatment with this inhibitor suppressed the cell proliferation in both K-Ras WT and mutated pancreatic cancer cells as well as PDX-C in a caspase-dependent manner. Conclusion: Our results identify SHP2 as a direct activator of Ras and potential therapeutic target for pancreatic cancer driven by a previously ‘undruggable’ oncogenic or hyperactive Ras. Citation Format: Yoshihito Kano, Severa Bunda, Jen Jen Yeh, Zhong-Yin Zhang, Michael Ohh.{Authors}. Novel treatment strategy for pancreatic cancer by targeting the ‘undruggable’ Ras oncoprotein. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B13.

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.

T1733 The Contribution of Atoh1/Hath1 to Phenotypic Gene Expression for Paneth Cells

with this activity. A combination of four subunits reached highest glucogenesis indicating a broader spectrum of glucosidase activities than any individual subunit alone. Some alphaLDx was not completely available for enzyme hydrolysis, and residues are considered either slowly digestible or resistant to human enzyme digestion. This study for the first time shows the direct role of individual mucosal glucosidase subunits in starch digestion and reveals the potential of producing slow glucose release dextrins.