79923

NANOG

We studied the expression and regulatory effects of ESC self-renewal molecule Nanog in colorectal cancer (CRC). Immunohistochemical analysis of 175 colorectal tumor samples showed that overexpression of Nanog was strongly correlated with poor prognosis, lymph node metastasis and Dukes classification for CRC. Univariate and multivariate survival analyses further indicated that Nanog expression was a potential prognostic factor for CRC. Gain-of-function analysis revealed that lentivirus-mediated Nanog overexpression promoted proliferation, motility and migration of human CRC cells. Interestingly, we found that Nanog played as both an inducer and a receipt of epithelial-mesenchymal transition (EMT) related signals. Nanog induced expression of Slug and Snail, two major regulator of EMT. Meanwhile, Nanog could also be regulated by Snail and initiated by TGF-beta1. Our data demonstrate self-renewal gene Nanog has a prognostic role in CRC, which functions in progression of CRC by promoting proliferation, invasion, and motility of human CRC cells, and participates EMT process during CRC progression.

Emerging evidence suggests that Nanog is involved in cervical tumorigenesis. However, the regulating role of Nanog in tumorigenesis and chemosensitivity are still poorly understood. In this study, Nanog was disrupted by transcription activator-like effector nucleases (TALEN) in Hela cells and its expression was significantly decreased in a single-cell derived sub-clone with biallelic mutations. The disruption of Nanog not only induced down regulation of some other core transcription factor genes for cell self-renewal, such as Oct4, Sox2 and FoxD3, but also led to the down regulation of some mesenchymal representative genes, vimentin and N-adherin, and up regulation of the epithelial gene, E-cadherin. In addition, the invasiveness and clonogenicity of the Hela cells were obviously affected, and surprisingly their sensitivities to anti-cancer drugs were also significantly increased in vitro. After Xenograft into nude mice, the growth volumes of the neoplasms from the Nanog disrupted Hela cells were significantly smaller compared with those from wild type ones. In conclusion, these results suggest that disruption of Nanog may reverse the status of epithelial-mesenchymal transition, which is critical in tumorigenesis, and alleviate chemoresistance, as well as their invasiveness, in cervical cancer cells.

Oct-4 and Nanog in regulating the epithelial-mesenchymal transition (EMT) and metastasis of breast cancer has not been clarified. We found that both Oct-4 and Nanog expression were significantly associated with tumor pathology and poor prognosis in 126 breast cancer patients. Characterization of CD44+CD24-cancer stem cell(CSC) derived from breast cancer cells indicated that CSC rapidly formed mammospheres and had potent tumorigenicity in vivo. Furthermore, TGF-beta up-regulated the expression of Oct-4, Nanog, N-cadherin, vimentin, Slug, and Snail, but down-regulated E-cadherin and cytokeratin 18 expression, demonstrating that CSC underwent EMT. Knockdown of both Oct-4 and Nanog expression inhibited spontaneous changes in the expression of EMT-related genes, while induction of both Oct-4 and Nanog over-expression enhanced spontaneous changes in the expression of EMT-related genes in CSC. However, perturbing alternation of Oct-4 and Nanog expression also modulated TGF-beta-induced EMT-related gene expression in CSC. Induction of Oct-4 and Nanog over-expression enhanced the invasiveness of CSC, but knockdown of both Oct-4 and Nanog inhibited the migration of CSC in vitro. Our data suggest that both Oct-4 and Nanog may serve as biomarkers for evaluating breast cancer prognosis. Our findings indicate that Oct-4 and Nanog positively regulate the EMT process, contributing to breast cancer metastasis.

BACKGROUND: Oct4 and Nanog are key regulatory genes that maintain the pluripotency and self-renewal properties of embryonic stem cells. We previously reported that the two stemness markers were tightly associated with cancer progression and poor outcomes of hepatocellular carcinoma. In this study, we demonstrate that coexpression of Oct4/Nanog modulates activation of signal transducer and activator of transcription 3 (Stat3), an oncogenic transcription factor that is activated in many human malignancies including hepatocellular carcinoma (HCC), as well as the expression of Snail, a key regulator implicated in epithelial-mesenchymal transition and tumor metastasis. METHODS: Oct4 and Nanog were ectopic expressed in MHCC97-L cell lines via lentiviral gene transfection. The stemness characteristics including self-renewal, proliferation, chemoresistance, and tumorigenicity were assessed. The effect of coexpression of Oct4 and Nanog on epithelial-mesenchymal transition change, and the underlying molecular signaling was investigated. RESULTS: Ectopic coexpression of Oct4 and Nanog empowered MHCC97-L cells with cancer stem cell (CSC) properties, including self-renewal, extensive proliferation, drug resistance, and high tumorigenic capacity. Significantly, Oct4 and Nanog encouraged epithelial-mesenchymal transition change contributing to tumor migration, invasion/metastasis in vitro and in vivo. Following molecular mechanism investigation indicated Oct4/Nanog-regulated epithelial-mesenchymal transition change through Stat3-dependent Snail activation. Moreover, silencing Stat3 abrogates Oct4/Nanog-mediated epithelial-mesenchymal transition (EMT) change and invasion/metastasis in HCC. CONCLUSIONS: We delineate Oct4 and Nanog initiate stem cell characteristics in hepatocellular carcinoma and promote epithelial-mesenchymal transition through activation of Stat3/Snail signaling. Our findings propose Stat3/Snail pathway as a novel therapeutic target for the treatment of progression and metastasis of HCC with CSC-like signatures and epithelial-mesenchymal transition phenotype.

Accumulating evidence indicates that Nanog plays a central role in modulating the biological behaviors of human hepatocellular carcinoma (HCC). However, the underlying mechanisms remain unclear. In the present study, we employed transcription activator-like effector nucleases (TALEN) to disrupt Nanog expression in HepG2 cells and obtained subcloned cells with diallelic Nanog mutations. Significantly, we found that the expression of pluripotency factors Sox2, Oct4 and Klf4, as well as expression of cancer stem cell (CSC) marker CD133, in the Nanog-targeted HepG2 cells was markedly downregulated. This finding suggests that Nanog may play an important role in maintaining the pluripotency and malignancy of HepG2 cells. We also revealed that Nanog regulated cell proliferation by modulating the expression of cyclin D1/D3/E1 and CDK2, respectively. Additionally, the disruption of Nanog resulted in the downregulation of epithelial-mesenchymal transition (EMT) regulators Snail and Twist, which contributed to the elevated level of epithelial marker E-cadherin, and to the decreased level of mesenchymal markers N-cadherin and vimentin in the HepG2 cells. In addition, the Nanog-targeted HepG2 cells exhibited reduced ability of invasion, migration and chemoresistance in vitro. In conclusion, the disruption of Nanog expression results in less proliferation, invasiveness, migration, more chemosensitivity and reversal of EMT in HepG2 cells, by which Nanog plays crucial roles in influencing the malignant phenotype of HepG2 cells.

NANOG is a key transcription factor that is overexpressed and plays an important role in various cancers. Its overexpression is associated with highly tumorigenic, drug-resistant, and poor prognosis. However, the underlying mechanism of action of NANOG in ovarian cancer remains unclear. epithelial-mesenchymal transition (EMT), which is a critical process in cancer invasion and metastasis, is also associated with drug resistance. We determined whether NANOG is associated with EMT and chemoresistance in epithelial ovarian cancer cells. NANOG expression was increased in epithelial ovarian cancer cells (HEY and SKOV3) compared with normal epithelial ovarian cells (Moody). Low expression of NANOG increased the expression of E-cadherin and decreased the expression of vimentin, beta-catenin, and Snail. Furthermore, the cell migration and invasion abilities were decreased. The multidrug resistance genes MDR-1 and GST-pi were also downregulated when NANOG was lowly expressed. The cells that were transfected with the si-NANOG plasmid were more sensitive to cisplatin compared with the cells that were transfected with empty vector. The data demonstrated that Stat3 was correlated with NANOG-mediated EMT and drug resistance. The silencing of Stat3 expression abrogated NANOG-mediated EMT changes and increased the sensitivity of the cells to chemotherapy. These results suggest that NANOG mediates EMT and drug resistance through activation of the Stat3 pathway in epithelial ovarian cancer.

BACKGROUND: Accumulating evidence supports the hypothesis that cancer stem cells (CSCs) are essential for cancer initiation, metastasis and drug resistance. However, the functional association of gastric CSC markers with stemness and epithelial-mesenchymal transition (EMT) signature genes is unclear. METHODS: qPCR was performed to measure the expression profiles of stemness and EMT signature genes and their association with putative CSC markers in gastric cancer tissues, cancer cell lines and sphere cells. Western blot analysis was used to confirm the results of the transcript analysis. Cell proliferation, cell migration, drug resistance and sphere cell growth assays were conducted to measure the expansion and invasion abilities of the cells. tumor xenograft experiments were performed in NOD/SCID mice to test cell stemness in vivo. Flow cytometry and immunofluorescence staining were used to analyze cell subpopulations. RESULTS: The expression of LGR5 was strikingly up-regulated in sphere cells but not in cancer tissues or parental adherent cells. The up-regulation of LGR5 was also positively associated with stemness regulators (NANOG, OCT4, SOX2, and AICDA) and EMT inducers (PRRX1, TWIST1, and BMI1). In addition, sphere cells exhibited up-regulated vimentin and down-regulated E-cadherin expression. Using gene-specific primers, we found that the NANOG expression primarily originates from the retrogene NANOGP8. Western blot analysis showed that the expression of both LGR5 and NANOG is significantly higher in sphere cells. LGR5 over-expression significantly enhanced sphere cell growth, cell proliferation, cell migration and drug resistance in MGC803 cells. tumor xenografts in nude mice showed that sphere cells are at least 10 times more efficient at tumor initiation than adherent cells. Flow cytometry analysis showed that ~20% of sphere cells are LGR5+/CD54+, but only ~3% of adherent cells are Lgr5+/CD54+. Immunofluorescence staining supports the above results. CONCLUSION: The LGR5-expressing fraction of CD54+ cells represents gastric cancer CSCs, in which LGR5 is closely associated with stemness and EMT core genes, and NANOG expression is mainly contributed by the retrogene NANOGP8. Sphere cells are the best starting materials for the characterization of CSCs.

Nanog is an important embryonic stem cell (ESC) gene that does not function as a classical oncogene, but needs to cooperate with other molecules to potentiate tumorigenic activity. The question addressed by the present study was whether a miRNA link exists between Nanog and epithelial-mesenchymal transition (EMT)-mesenchymal-epithelial transition (MET) plasticity. Here, we found that Nanog mRNA expression level was inversely correlated with miR-200c and miR-200b expression levels in colon cancer cell lines and human colorectal cancer tissues. Forced Nanog expression in low-Nanog colon cancer cells inhibited miR-200c and miR-200b expression, and interfered Nanog expression in high-Nanog colon cancer cells promoted miR-200c and miR-200b expression. Furthermore, we confirmed that Nanog directly repressed transcription of the miR-200c and miR-200b genes, and miR-200c and miR-200b mediated Nanog-induced EMT occurrence. Luciferase and ChIP assays determined that Nanog bound directly to the potential Nanog binding sites in the miR-200c and miR-200b promoters and repressed their transcription. In conclusion, our findings suggest that Nanog modulates EMT-MET plasticity by regulating miR-200 clusters via a direct transcriptional mechanism, and the Nanog-miR-200 axis may be a good therapeutic target for CRC control.CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.