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NANOGP8 is the key regulator of stemness, EMT, Wnt pathway, chemoresistance, and other malignant phenotypes in gastric cancer cells.

ABSTRACT: Accumulating evidence demonstrated that NANOG1, the key transcription factor for embryonic stem cells, is associated with human cancers. NANOGP8, one of the pseudogenes in NANOG gene family, contains an intact open reading frame and also said to be expressed in cancer tissues. Therefore, a systematic study is greatly needed to address the following questions: among NANOG1 and NANOGP8, which gene is the main contributor for NANOG expression in cancer cells and which one is the key regulator responsible for stemness, epithelial-mesenchymal transition (EMT), metastasis, chemoresistance and other malignant phenotypes. Here we try to explore these issues with gastric adenocarcinoma cell lines in vitro using variety of molecular and cellular techniques.Special primers were designed to distinguish PCR products from NANOG1 and NANOGP8. Sphere-forming cells were cultured with serum-free and selective medium. A stable cell line was established with infection of lentivirus containing NANOGP8. qPCR was performed to measure NANOGP8 expression and its association with stemness, EMT and CSC markers in adherent cells and sphere-forming cells. Western blot analysis was deployed to confirm results of the transcript analysis. Experiments of cell proliferation, migration, invasion, clonogenic assay, sphere cell growth assays, cell cycle analysis, ?-catenin accumulation and translocation in nucleus, and drug resistance were conducted to measure the impact of NANOGP8 on malignant statuses of gastric cancer cells. Immunofluorescence staining was used to analyze cell subpopulations with different markers.NANOGP8 is mainly responsible for NANOG expression in sphere-forming (stem cell-like) cells derived from gastric cancer cell lines regardless their differentiation status. Ectopic expression of NANOGP8 significantly up-regulates stemness transcription factors, EMT inducers, and cancer stem cell markers (CSC) including Lgr5. NANOGP8 also promotes expression of the signature genes vimentin and N-caderin for mesenchymal cells and down-regulates the signature gene E-caderin for epithelial cells whereby confer the cells with mesenchymal cell phenotype. In NANOGP8 over-expressed adherent and sphere-forming cells, Lgr5+ cells are significantly increased. Ectopic expression of NANOGP8 endows gastric cells with enhanced proliferation, migration, invasion, sphere-forming and clonogenic capacity, and chemoresistance. NANOGP8 expression also enhances ?-catenin accumulation in nucleus and strengthens Wnt signal transduction.NANOGP8 is the main regulator of gastric cancer stem cells. It is closely associated with EMT, stemness, and CSC marker as well as Wnt signal pathway. NANOGP8 is correlated with cell proliferation, migration, invasion, clonogenic capacity, ?-catenin accumulation in nucleus, and chemoresistance in gastric cancer. NANOGP8 is a promising molecular target for clinical intervention of gastric cancer.

SUBMITTER: Ma X

PROVIDER: S-EPMC5915267 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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NANOGP8 is the key regulator of stemness, EMT, Wnt pathway, chemoresistance, and other malignant phenotypes in gastric cancer cells.

Ma Xia X Wang Bei B Wang Xiaofang X Luo Yujiao Y Fan Wufang W

PloS one 20180424 4

<h4>Background</h4>Accumulating evidence demonstrated that NANOG1, the key transcription factor for embryonic stem cells, is associated with human cancers. NANOGP8, one of the pseudogenes in NANOG gene family, contains an intact open reading frame and also said to be expressed in cancer tissues. Therefore, a systematic study is greatly needed to address the following questions: among NANOG1 and NANOGP8, which gene is the main contributor for NANOG expression in cancer cells and which one is the ...[more]

PMID: 29689047

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Project description: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.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.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.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.

Project description:BACKGROUND:As the most abundant epigenetic modification on mRNAs and long non-coding RNAs, N6-methyladenosine (m6A) modification extensively exists in mammalian cells. Controlled by writers (methyltransferases), readers (signal transducers), and erasers (demethylases), m6A influences mRNA structure, maturation, and stability, thus negatively regulating protein expression in a post-translational manner. Nevertheless, current understanding of m6A's roles in tumorigenesis, especially in gastric cancer (GC) remains to be unveiled. In this study, we assessed m6A's clinicopathological relevance to GC and explored the underlying mechanisms. METHODS:By referring to a proteomics-based GC cohort we previously generated and the TCGA-GC cohort, we merged expressions of canonical m6A writers (METTL3/METTL14), readers (YTHDF1/YTHDF2/YTHDF3), and erasers (ALKBH5/FTO), respectively, as W, R, and E signatures to represent m6A modification. We stratified patients according to these signatures to decipher m6A's associations with crucial mutations, prognosis, and clinical indexes. m6A's biological functions in GC were predicted by gene set enrichment analysis (GSEA) and validated by in vitro experiments. RESULTS:We discovered that W and R were potential tumor suppressive signatures, while E was a potential oncogenic signature in GC. According to W/R/E stratifications, patients with low m6A-indications were accompanied with higher mutations of specific genes (CDH1, AR, GLI3, SETBP1, RHOA, MUC6, and TP53) and also demonstrated adverse clinical outcomes. GSEA suggested that reduced m6A was correlated with oncogenic signaling and phenotypes. Through in vitro experiments, we proved that m6A suppression (represented by METTL14 knockdown) promoted GC cell proliferation and invasiveness through activating Wnt and PI3K-Akt signaling, while m6A elevation (represented by FTO knockdown) reversed these phenotypical and molecular changes. m6A may also be involved in interferon signaling and immune responses of GC. CONCLUSIONS:Our work demonstrated that low-m6A signatures predicted adverse clinicopathological features of GC, while the reduction of RNA m6A methylation activated oncogenic Wnt/PI3K-Akt signaling and promoted malignant phenotypes of GC cells.

Project description:BACKGROUND:Transmembrane 4?L six family member 1 (TM4SF1) is upregulated in several epithelial cancers and is closely associated with poor prognosis. However, the role of TM4SF1 and its potential mechanism in colorectal cancer (CRC) remain elusive. METHODS:We investigated the expression of TM4SF1 in the Oncomine, the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and confirmed the results by immunohistochemistry (IHC), qPCR and Western blotting (WB) of CRC tissues. The effect of TM4SF1 on the epithelial-to-mesenchymal transition (EMT) and cancer stemness of CRC cells was investigated by Transwell, wound healing and sphere formation assays. A series of in vitro and in vivo experiments were conducted to reveal the mechanisms by which TM4SF1 modulates EMT and cancer stemness in CRC. RESULTS:TM4SF1 expression was markedly higher in CRC tissues than in non-tumour tissues and was positively correlated with poor prognosis. Downregulation of TM4SF1 inhibited the migration, invasion and tumour sphere formation of SW480 and LoVo cells. Conversely, TM4SF1 overexpression significantly enhanced the migration, invasion and tumoursphere formation potential of CRC cells, Additionally, TM4SF1 silencing inhibited the EMT mediated by transforming growth factor-?1 (TGF-?1). Mechanistically, gene set enrichment analysis (GSEA) predicted that the Wnt signalling pathway was one of the most impaired pathways in TM4SF1-deficient CRC cells compared to controls. The results were further validated by WB, which revealed that TM4SF1 modulated SOX2 expression in a Wnt/?-catenin activation-dependent manner. Furthermore, we found that knockdown of TM4SF1 suppressed the expression of c-Myc, leading to decreased c-Myc binding to the SOX2 gene promoter. Finally, depletion of TM4SF1 inhibited metastasis and tumour growth in a xenograft mouse model. CONCLUSION:Our study substantiates a novel mechanism by which TM4SF1 maintains cancer cell stemness and EMT via the Wnt/?-catenin/c-Myc/SOX2 axis during the recurrence and metastasis of CRC.

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NANOGP8 is the key regulator of stemness, EMT, Wnt pathway, chemoresistance, and other malignant phenotypes in gastric cancer cells.

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NANOGP8 is the key regulator of stemness, EMT, Wnt pathway, chemoresistance, and other malignant phenotypes in gastric cancer cells.

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