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Tu2068 The Ribonuclease RNaseH2b Controls Intestinal Stem Cell Integrity
Tu2067 CD133-Targeted Oncolytic Adenovirus Shows Therapeutic Effect By attacking Colon Cancer Stem Cells Mizuho Sato-Dahlman, Yoshiaki Miura, Masato Yamamoto
AGA Abstracts
Colorectal cancer is the third most common cancer in the world and about 50% of patients relapse after treatment. Cancer stem cells (CSCs) have contribution to recurrence, metastasis and and chemotherapy resistant of colorectal cancers. Therefore, eliminating CSCs is a key approach to improve colorectal cancer treatment. CD133 (Prominin-1), a member of the transmembrane glycoprotein family, is a marker of CSCs in several cancers and is also generally accepted as a colorectal cancer stem cell marker. CD133 expression was correlated with recurrence, metastases and chemotherapy resistance, as well as poor prognosis in colorectal cancer. Recently, we have established a method for isolating transductionallytargeted adenovirus by high-throughput screening. In this study, using this adenovirus library screening system, we isolated the CD133-specific Oncolytic Adenovirus (OAd) and tested the oncolytic activity of CD133-targeted Ad in both in vitro and in vivo. CD133targeted OAd (AdML-TYML) showed strong binding to CD133-positive cells (293-CD133 and LoVo (CD133 (+) human colon carcinoma)), not to CD133-negative cells (parental 293 and LS174T (CD133 (-) human colon carcinoma)) and this virus also showed strong oncolysis selectively in LoVo cells, whereas there was no effect on LS174T cells. In order to assess the effect of AdML-TYML on stemness, tumor establishment assay was performed. AdMLTYML treatment leads to inhibition of tumor establishment of CD133-positive colorectal cancer cell lines in nude mice . 100% of the mice inoculated with non-infected LoVo cells had developed tumors, while 0% of the mice inoculated with AdML-TYML infected LoVo cells had done so. In addition, when the anti-tumor effect of the CD133-targeted OAd was analyzed in established tumors of CD133(+) colorectal cancer subcutaneous xenografts, intra-tumor (i.t.) administration of AdML-TYML exhibited significantly stronger antitumor effect compared to its equivalent Ad5-WT (wild type Ad5 fiber). Our results suggest that CD133-targeted oncolytic adenovirus selectively eliminate CD133 (+) colorectal cancer stem cells from the tumor and also this virus could be a key tool for the prevention of metastases and relapses in a variety of cancers.
Tu2069 ATOH1 Protein Stabilization in Tumor Acquires the Morphological Change to Signet Ring Cell Carcinoma With Cancer Stem Cell Enrichment in Colon Cancer Kiichiro Tsuchiya, Shuji Hibiya, Keita Fukushima, Tomoaki Shirasaki, Ryuichi Okamoto, Tetsuya Nakamura, Mamoru Watanabe Background: Atonal homolog 1 (Atoh1) is essential for the differentiation toward secretory lineages of intestinal epithelial cells. We have reported that Atoh1 protein in sporadic colon cancer was degraded by APC deletion in Wnt signal using proteasomal system, resulting in non-mucinous cancer (Gastroenterol. 2007). While, we have recently found that Atoh1 protein was expressed in mucinous cancer of the patients with ulcerative colitis. Moreover, TNF-a stabilized Atoh1 protein, resulting in the acquisition of mucinous form of colitis-associated cancer with more malignant potential (Cancer Sci. 2015). The relationship between mucinous form and malignant potential including cancer stem cell regulation, however, has not been elucidated. In this study, we therefore aimed to assess the behavior of cancer stem cells in Atoh1 positive mucinous tumor. Methods: The mCherryAtoh1 vector was generated by inserting Atoh1 gene fused with mCherry gene. The Atoh1 mutant (mCherry 5SA-Atoh1) was constructed by the replacement of five serine residues with alanines to stabilize its protein in sporadic human colon cancer-derived DLD1 cells (BBRC 2013). Human Lgr5 reporter plasmid was generated by cloning a 1000bp sequence 5' of the human Lgr5 promoter region into a GFP vector. Each vector was transfected into DLD1 cells. Tumor was generated by inoculation of DLD1 cells into nude mice. Pathological finding of each tumor was assessed by HE and Alcian blue staining. Tumorigenesis was assessed by Xenograft formation assay with limiting dilution transplantation. Lgr5 expression was assessed by RT-PCR and flow cytoetry. Results: mCherry-5SA-Atoh1 protein was stably expressed in DLD1 cells whereas mCherry-Atoh1 protein was degraded in DLD1 cells. Xenograft tumor was generated by the inoculation of either mCherry-Atoh1/DLD1 cells or mCherry-5SA-Atoh1/DLD1 cells into nude mice for 20days. Alcian blue staining showed mucinous and signet ring shaped cancer cells only in tumors derived from mCherry-5SAAtoh1/DLD1 cells. Cancer stem cells were enriched in 5SA-Atoh1 tumor with Lgr5 upregulation. In addition, Lgr5 promoter GFP was therefore transfected into mCherry-Atoh1/DLD1 cells and mCherry-5SA-Atoh1/DLD1 cells by lentiviral vector system, respectively. Strength of GFP fluorescence was related to the expression of Lgr5 in tumor. Tumor cells were divided with GFP high and low cells only in 5SA-Atoh1 tumor. Conclusion: Atoh1 expression in tumor might be crucial for the signet ring shape formation and mucinous phenotype with the enrichment of cancer stem cells. Moreover Atoh1 might promote cancer stem cell niche in tumor.
Tu2068 The Ribonuclease RNaseH2b Controls Intestinal Stem Cell Integrity Konrad Aden, Kareen Bartsch, Florian Tran, Stefan Rose John, Philip Rosenstiel, Björn Rabe Background: The stability of genomic DNA is under a tightly controlled surveillance. Especially in high proliferating cells, as e.g. intestinal stem cells, RNA/DNA hybrids display a menace to DNA integritiy. The ribonuclease RNAseH2b removes RNA/DNA hybrids and thereby ensures cellular proliferation. Hypomoprhic mutations of the RNAseH2b gene are associated with Aicardi-Goutières syndrome that results in a spontaneous inflammatory phenotype. We tested the role of RNAseH2b in maintaining proliferation and regeneration in the intestinal epithelium Methods: We generated RNAseH2bfl/fl and RNAseH2bDIEC to study the role of RNAseH2b in the intestinal epithelium. WB, RT-PCR and IHC were performed to study the basal phenotype of unchallenged WT and KO mice. Acute DSS colitis was induced to investigate the impact of RNAseH2b on intestinal regeneration. AOMDSS colitis was induced to study the role of RNAseH2b on intestinal carcinogenesis. Organoids of RNAseH2bfl/fl and RNAseH2bDIEC were subjected to RNA sequencing. Results: No macromorphological difference was seen between RNAseH2bfl/fl and RNAseH2bDIEC, with respect to age dependent body weight gain. Histological characterization reveals spontaneous DNA double strand breaks (DSB) in epithelial crypts of RNAseH2bDIEC, which leads to a restriction of epithelial stemness, as measured by expression of stem cell markers (Olfm4, Lgr5). When mice were challenged to acute DSS colitis, RNAseH2bDIEC mice reveal a strong phenotype with dramatic weight loss, increased histological disease activity and impaired intestinal regeneration. Interestingly, when mice were challenged to AOM-DSS colitis, mice again showed increased intestinal inflammation but developed significantly less tumors. Decreased tumor development was due to DNA induced cellular senescence, as shown by acid bgalactosidase staining in intestinal crypts in RNAseH2bDIEC but not RNAseH2bfl/fl mice. Conclusion: We show for the first time, that the RNAseH2b plays an essential role in maintaining intestinal regeneration by protecting genomic DNA of high proliferating cells from DNA/RNA hybrids induced DNA damage. Knockout of RNAseH2b leads to loss of epithelial stemness and induction of cellular senescence. Keywords: DNA Damage, Stem Cell, AOM DSS, Intestinal Inflammation
Tu2070 Fas Signaling Induces Stemness Properties in Colorectal Cancer Haoxuan Zheng Fas signaling promotes colorectal cancer(CRC) metastasis by inducing epithelial- mesenchymal transition (EMT). Acquisition of EMT propreties in turn induces stem cell-like properties. However, the mechanism by which Fas signaling contributes to stemness in CRC still remains unclear. Hence, the aim of this study was to investigate how Fas signaling regulates CRC stemness. For this purpose soft agar assay, sphere formation assay, cell survival analysis, immunoblot, qRT-PCR, chromatin immunoprecipitation, and luciferase reporter assay were performed. FasL, Bmi1 and miR-200c expression in human CRC specimens was examined by immunohistochemistry, qRT-PCR and immunoblot .We showed that Fas signaling induces stem cell properties in CRC, relying on ERK1/2 MAPK pathway and that Bmi1 was mainly responsible for FasL-induced stemness. FasL treatment promoted Bmi1 expression by inhibiting miR-200c that targets Bmi1 3'UTR. Furthermore, FasL-induced Zeb1 binded with miR200c promoter and inhibited its expression. Moreover, FasL-induced b-catenin nuclear expression promoted Zeb1 expression by binding with Zeb1 promoter. GSK-3 b which regulates b-catenin was inhibited by FasL-induced ERK1/2 MAPK signaling. Finally, FasL and Bmi1 expression in clinical samples increased during CRC progression and a positive correlation between them was observed. Patients with high FasL and Bmi1 expression had worse prognosis compared to patients with low expression. In conclusion, our results showed that Fas signaling can promote stemness in CRC through the modulation of Bmi1 expression via ERK1/2 MAPK/GSK3b/b-catenin/Zeb1/miR-200c axis, suggesting that Fas signaling- based cancer therapies should be administered cautiously, as activation of this pathway not only leads to apoptosis but also induces stemness in CRC.
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AGA Abstracts
AGA Abstracts
Colorectal cancer is the third most common cancer in the world and about 50% of patients relapse after treatment. Cancer stem cells (CSCs) have contribution to recurrence, metastasis and and chemotherapy resistant of colorectal cancers. Therefore, eliminating CSCs is a key approach to improve colorectal cancer treatment. CD133 (Prominin-1), a member of the transmembrane glycoprotein family, is a marker of CSCs in several cancers and is also generally accepted as a colorectal cancer stem cell marker. CD133 expression was correlated with recurrence, metastases and chemotherapy resistance, as well as poor prognosis in colorectal cancer. Recently, we have established a method for isolating transductionallytargeted adenovirus by high-throughput screening. In this study, using this adenovirus library screening system, we isolated the CD133-specific Oncolytic Adenovirus (OAd) and tested the oncolytic activity of CD133-targeted Ad in both in vitro and in vivo. CD133targeted OAd (AdML-TYML) showed strong binding to CD133-positive cells (293-CD133 and LoVo (CD133 (+) human colon carcinoma)), not to CD133-negative cells (parental 293 and LS174T (CD133 (-) human colon carcinoma)) and this virus also showed strong oncolysis selectively in LoVo cells, whereas there was no effect on LS174T cells. In order to assess the effect of AdML-TYML on stemness, tumor establishment assay was performed. AdMLTYML treatment leads to inhibition of tumor establishment of CD133-positive colorectal cancer cell lines in nude mice . 100% of the mice inoculated with non-infected LoVo cells had developed tumors, while 0% of the mice inoculated with AdML-TYML infected LoVo cells had done so. In addition, when the anti-tumor effect of the CD133-targeted OAd was analyzed in established tumors of CD133(+) colorectal cancer subcutaneous xenografts, intra-tumor (i.t.) administration of AdML-TYML exhibited significantly stronger antitumor effect compared to its equivalent Ad5-WT (wild type Ad5 fiber). Our results suggest that CD133-targeted oncolytic adenovirus selectively eliminate CD133 (+) colorectal cancer stem cells from the tumor and also this virus could be a key tool for the prevention of metastases and relapses in a variety of cancers.
Tu2069 ATOH1 Protein Stabilization in Tumor Acquires the Morphological Change to Signet Ring Cell Carcinoma With Cancer Stem Cell Enrichment in Colon Cancer Kiichiro Tsuchiya, Shuji Hibiya, Keita Fukushima, Tomoaki Shirasaki, Ryuichi Okamoto, Tetsuya Nakamura, Mamoru Watanabe Background: Atonal homolog 1 (Atoh1) is essential for the differentiation toward secretory lineages of intestinal epithelial cells. We have reported that Atoh1 protein in sporadic colon cancer was degraded by APC deletion in Wnt signal using proteasomal system, resulting in non-mucinous cancer (Gastroenterol. 2007). While, we have recently found that Atoh1 protein was expressed in mucinous cancer of the patients with ulcerative colitis. Moreover, TNF-a stabilized Atoh1 protein, resulting in the acquisition of mucinous form of colitis-associated cancer with more malignant potential (Cancer Sci. 2015). The relationship between mucinous form and malignant potential including cancer stem cell regulation, however, has not been elucidated. In this study, we therefore aimed to assess the behavior of cancer stem cells in Atoh1 positive mucinous tumor. Methods: The mCherryAtoh1 vector was generated by inserting Atoh1 gene fused with mCherry gene. The Atoh1 mutant (mCherry 5SA-Atoh1) was constructed by the replacement of five serine residues with alanines to stabilize its protein in sporadic human colon cancer-derived DLD1 cells (BBRC 2013). Human Lgr5 reporter plasmid was generated by cloning a 1000bp sequence 5' of the human Lgr5 promoter region into a GFP vector. Each vector was transfected into DLD1 cells. Tumor was generated by inoculation of DLD1 cells into nude mice. Pathological finding of each tumor was assessed by HE and Alcian blue staining. Tumorigenesis was assessed by Xenograft formation assay with limiting dilution transplantation. Lgr5 expression was assessed by RT-PCR and flow cytoetry. Results: mCherry-5SA-Atoh1 protein was stably expressed in DLD1 cells whereas mCherry-Atoh1 protein was degraded in DLD1 cells. Xenograft tumor was generated by the inoculation of either mCherry-Atoh1/DLD1 cells or mCherry-5SA-Atoh1/DLD1 cells into nude mice for 20days. Alcian blue staining showed mucinous and signet ring shaped cancer cells only in tumors derived from mCherry-5SAAtoh1/DLD1 cells. Cancer stem cells were enriched in 5SA-Atoh1 tumor with Lgr5 upregulation. In addition, Lgr5 promoter GFP was therefore transfected into mCherry-Atoh1/DLD1 cells and mCherry-5SA-Atoh1/DLD1 cells by lentiviral vector system, respectively. Strength of GFP fluorescence was related to the expression of Lgr5 in tumor. Tumor cells were divided with GFP high and low cells only in 5SA-Atoh1 tumor. Conclusion: Atoh1 expression in tumor might be crucial for the signet ring shape formation and mucinous phenotype with the enrichment of cancer stem cells. Moreover Atoh1 might promote cancer stem cell niche in tumor.
Tu2068 The Ribonuclease RNaseH2b Controls Intestinal Stem Cell Integrity Konrad Aden, Kareen Bartsch, Florian Tran, Stefan Rose John, Philip Rosenstiel, Björn Rabe Background: The stability of genomic DNA is under a tightly controlled surveillance. Especially in high proliferating cells, as e.g. intestinal stem cells, RNA/DNA hybrids display a menace to DNA integritiy. The ribonuclease RNAseH2b removes RNA/DNA hybrids and thereby ensures cellular proliferation. Hypomoprhic mutations of the RNAseH2b gene are associated with Aicardi-Goutières syndrome that results in a spontaneous inflammatory phenotype. We tested the role of RNAseH2b in maintaining proliferation and regeneration in the intestinal epithelium Methods: We generated RNAseH2bfl/fl and RNAseH2bDIEC to study the role of RNAseH2b in the intestinal epithelium. WB, RT-PCR and IHC were performed to study the basal phenotype of unchallenged WT and KO mice. Acute DSS colitis was induced to investigate the impact of RNAseH2b on intestinal regeneration. AOMDSS colitis was induced to study the role of RNAseH2b on intestinal carcinogenesis. Organoids of RNAseH2bfl/fl and RNAseH2bDIEC were subjected to RNA sequencing. Results: No macromorphological difference was seen between RNAseH2bfl/fl and RNAseH2bDIEC, with respect to age dependent body weight gain. Histological characterization reveals spontaneous DNA double strand breaks (DSB) in epithelial crypts of RNAseH2bDIEC, which leads to a restriction of epithelial stemness, as measured by expression of stem cell markers (Olfm4, Lgr5). When mice were challenged to acute DSS colitis, RNAseH2bDIEC mice reveal a strong phenotype with dramatic weight loss, increased histological disease activity and impaired intestinal regeneration. Interestingly, when mice were challenged to AOM-DSS colitis, mice again showed increased intestinal inflammation but developed significantly less tumors. Decreased tumor development was due to DNA induced cellular senescence, as shown by acid bgalactosidase staining in intestinal crypts in RNAseH2bDIEC but not RNAseH2bfl/fl mice. Conclusion: We show for the first time, that the RNAseH2b plays an essential role in maintaining intestinal regeneration by protecting genomic DNA of high proliferating cells from DNA/RNA hybrids induced DNA damage. Knockout of RNAseH2b leads to loss of epithelial stemness and induction of cellular senescence. Keywords: DNA Damage, Stem Cell, AOM DSS, Intestinal Inflammation
Tu2070 Fas Signaling Induces Stemness Properties in Colorectal Cancer Haoxuan Zheng Fas signaling promotes colorectal cancer(CRC) metastasis by inducing epithelial- mesenchymal transition (EMT). Acquisition of EMT propreties in turn induces stem cell-like properties. However, the mechanism by which Fas signaling contributes to stemness in CRC still remains unclear. Hence, the aim of this study was to investigate how Fas signaling regulates CRC stemness. For this purpose soft agar assay, sphere formation assay, cell survival analysis, immunoblot, qRT-PCR, chromatin immunoprecipitation, and luciferase reporter assay were performed. FasL, Bmi1 and miR-200c expression in human CRC specimens was examined by immunohistochemistry, qRT-PCR and immunoblot .We showed that Fas signaling induces stem cell properties in CRC, relying on ERK1/2 MAPK pathway and that Bmi1 was mainly responsible for FasL-induced stemness. FasL treatment promoted Bmi1 expression by inhibiting miR-200c that targets Bmi1 3'UTR. Furthermore, FasL-induced Zeb1 binded with miR200c promoter and inhibited its expression. Moreover, FasL-induced b-catenin nuclear expression promoted Zeb1 expression by binding with Zeb1 promoter. GSK-3 b which regulates b-catenin was inhibited by FasL-induced ERK1/2 MAPK signaling. Finally, FasL and Bmi1 expression in clinical samples increased during CRC progression and a positive correlation between them was observed. Patients with high FasL and Bmi1 expression had worse prognosis compared to patients with low expression. In conclusion, our results showed that Fas signaling can promote stemness in CRC through the modulation of Bmi1 expression via ERK1/2 MAPK/GSK3b/b-catenin/Zeb1/miR-200c axis, suggesting that Fas signaling- based cancer therapies should be administered cautiously, as activation of this pathway not only leads to apoptosis but also induces stemness in CRC.
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AGA Abstracts