Limited sorafenib anticancer effects on primary cultured hepatocellular carcinoma cells with high NANOG expression.

Cancer stem cell is considered as an important cause to exacerbate the prognosis. NANOG and POU5F1 are markers for cancer stem cells. The associations between NANOG and POU5F1 expressions with the sorafenib anticancer effects in primary cultured hepatocellular carcinoma (HCC) cells were investigated. Eight primary cultured HCC parent cell lines and 13 subgroups established by flow cytometric sorting using NANOG and POU5F1 as targets were investigated with clinically achievable sorafenib plasma concentrations (5 and 10 µg/mL). Sorafenib showed obvious downregulation of RAF/MEK/ERK signaling pathways and dose-dependent anti-proliferative effects only on s003 parent cell line, which showed the lowest expression of NANOG among all tested cell lines except one downregulated NANOG with upregulated POU5F1 s020 subgroup. Sorafenib also inhibited proliferation in this s020 subgroup but promoted proliferation in its parent cell line. For the only one downregulated NANOG alone s015 subgroup, sorafenib which had no influence on its parent cell line inhibited proliferation in this subgroup. Only the above three cell lines could demonstrate sorafenib antiproliferative effects. On the contrary, sorafenib promoted proliferation in three (s003, s015, s071) out of four upregulated NANOG alone subgroups. On the other hand, Sorafenib showed diverse influence on proliferation among four upregulated POU5F1 alone subgroups. In conclusion, NANOG rather than POU5F1 expression is a critical marker for the anticancer effects of sorafenib on HCC. The sorafenib anticancer effects on HCC cells with high NANOG expression were limited. Sorafenib should be combined with other drug able to target cancer cells with high NANOG expression.

TNF-α up-regulates Nanog by activating NF-κB pathway to induce primary rat spinal cord astrocytes dedifferentiation.

AIMS: Astrocytes re-acquire stem cell potential upon inflammation, thereby becoming a promising source of cells for regenerative medicine. Nanog is an essential transcription factor to maintain the characteristics of stem cells. We aimed to investigate the role of Nanog in astrocyte dedifferentiation. MAIN METHODS: TNF-α was used to induce the dedifferentiation of primary rat spinal cord astrocytes. The expression of immature markers CD44 and Musashi-1 was detected by qRT-PCR and immunofluorescence. The Nanog gene is knocked down by small interference RNA. Nanog expression was measured by qRT-PCR and western blotting. BAY 11-7082 was used to suppress NF-κB signals in astrocytes. NF-κB signaling was evaluated by Western blotting. KEY FINDINGS: Our results showed that TNF-α promoted the re-expression of CD44 and Musashi-1 in astrocytes. Dedifferentiated astrocytes could be induced to differentiate into oligodendrocyte lineage cells indicating that the astrocytes had pluripotency. In addition, TNF-α treatment activated NF-κB signaling pathway and up-regulated Nanog. Knockdown of Nanog reversed the increase of CD44 and Musashi-1 induced by TNF-α without affecting the activation of NF-κB signaling. Importantly, blocking NF-κB signaling by BAY 11-7082 inhibited the expression of immature markers suggesting that TNF-α induces dedifferentiation of astrocytes through the NF-κB signaling pathway. BAY 11-7082 could also inhibit the expression of Nanog, which indicated that Nanog was regulated by NF-κB signaling pathway. SIGNIFICANCE: These findings indicate that activation of the NF-κB signaling pathway through TNF-α leads to astrocytes dedifferentiation via Nanog. These results expand our understanding of the mechanism of astrocytes dedifferentiation.

Warburg effect-promoted exosomal circ_0072083 releasing up-regulates NANGO expression through multiple pathways and enhances temozolomide resistance in glioma.

BACKGROUND: Temozolomide (TMZ) resistance limits its application in glioma. Exosome can carry circular RNAs (circRNAs) to regulate drug resistance via sponging microRNAs (miRNAs). miRNAs can control mRNA expression by regulate the interaction with 3'UTR and methylation. Nanog homeobox (NANOG) is an important biomarker for TMZ resistance. Hitherto, it is unknown about the role of exosomal hsa_circ_0072083 (circ_0072083) in TMZ resistance in glioma, and whether it is associated with NANOG via regulating miRNA sponge and methylation. METHODS: TMZ-resistant (n = 36) and sensitive (n = 33) patients were recruited. The sensitive cells and constructed resistant cells were cultured and exposed to TMZ. circ_0072083, miR-1252-5p, AlkB homolog H5 (ALKBH5) and NANOG levels were examined via quantitative reverse transcription polymerase chain reaction and western blot. The half maximal inhibitory concentration (IC50) of TMZ, cell proliferation, apoptosis, migration and invasion were analyzed via Cell Counting Kit-8, colony formation, flow cytometry, wound healing and transwell assays. The in vivo function was assessed using xenograft model. The N6-methyladenosine (m6A) level was analyzed via methylated RNA immunoprecipitation (MeRIP). Target relationship was investigated via dual-luciferase reporter assay and RNA immunoprecipitation. Warburg effect was investigated via lactate production, glucose uptake and key enzymes expression. Exosome was isolated and confirmed via transmission electron microscopy and specific protein expression. RESULTS: circ_0072083 expression was increased in TMZ-resistant glioma tissues and cells. circ_0072083 knockdown restrained the resistance of resistant cells via decreasing IC50 of TMZ, proliferation, migration, invasion and xenograft tumor growth and increasing apoptosis. circ_0072083 silence reduced NANOG expression via blocking ALKBH5-mediated demethylation. circ_0072083 could regulate NANOG and ALKBH5 via targeting miR-1252-5p to control TMZ resistance. Warburg effect promoted the release of exosomal circ_0072083 in resistant cells. Exosomal circ_0072083 from resistant cells increased the resistance of sensitive cells to TMZ in vitro and xenograft model. Exosomal circ_0072083 level was enhanced in resistant patients, and it had a diagnostic value and indicated a lower overall survival in glioma. CONCLUSION: Exosomal circ_0072083 promoted TMZ resistance via increasing NANOG via regulating miR-1252-5p-mediated degradation and demethylation in glioma.

Effect of Nanog overexpression on the metastatic potential of a mouse melanoma cell line B16-BL6.

Nanog, a marker and regulator of the undifferentiated state in embryonic stem cells were anticipated to be an effective enhancer of cancer metastasis. We have developed a Nanog overexpressing mouse melanoma cell line B16-BL6 (BL6). BL6 was well recognized as a cell line with a high metastatic potential. In vitro tests revealed the enhancement of cell proliferation, wound healing activity, and matrix metalloproteinase 9 (MMP9) activity. Nanog-induced up- or down-regulated genes were comprehensively analyzed by transcriptome sequencing using Nanog+BL6 and wild-type BL6. Principally, up-regulated genes were involved in vesicle-aided glucose transport and oxidative phosphorylation, while down-regulated genes were associated with immunosuppression and apoptosis. A marked finding was that TGF-ß1 was down-regulated, because TGF-ß1 has been well discussed about its suppressive/progressive dual role in cancer. In vivo test showed that the number and volume of metastatic colonies of BL6 to lung were as high as 115 colonies/lung and 5.6 mm3/lung. Under this condition, Nanog overexpression caused a progressive effect (150 colonies/lung, p = 0.25; 9.2 mm3/lung, p = 0.13) rather than a suppressive effect on the metastasis. In this study, the effectiveness of Nanog overexpression in enhancing the metastatic potential of melanoma cell lines has been demonstrated for the first time.

NANOG Attenuates Hair Follicle-Derived Mesenchymal Stem Cell Senescence by Upregulating PBX1 and Activating AKT Signaling.

Stem cells derived from elderly donors or harvested by repeated subculture exhibit a marked decrease in proliferative capacity and multipotency, which not only compromises their therapeutic potential but also raises safety concerns for regenerative medicine. NANOG-a well-known core transcription factor-plays an important role in maintaining the self-renewal and pluripotency of stem cells. Unfortunately, the mechanism that NANOG delays mesenchymal stem cell (MSC) senescence is not well-known until now. In our study, we showed that both ectopic NANOG expression and PBX1 overexpression (i) significantly upregulated phosphorylated AKT (p-AKT) and PARP1; (ii) promoted cell proliferation, cell cycle progression, and osteogenesis; (iii) reduced the number of senescence-associated-ß-galactosidase- (SA-ß-gal-) positive cells; and (iv) downregulated the expression of p16, p53, and p21. Western blotting and dual-luciferase activity assays showed that ectopic NANOG expression significantly upregulated PBX1 expression and increased PBX1 promoter activity. In contrast, PBX1 knockdown by RNA interference in hair follicle- (HF-) derived MSCs that were ectopically expressing NANOG resulted in the significant downregulation of p-AKT and the upregulation of p16 and p21. Moreover, blocking AKT with the PI3K/AKT inhibitor LY294002 or knocking down AKT via RNA interference significantly decreased PBX1 expression, while increasing p16 and p21 expression and the number of SA-ß-gal-positive cells. In conclusion, our findings show that NANOG delays HF-MSC senescence by upregulating PBX1 and activating AKT signaling and that a feedback loop likely exists between PBX1 and AKT signaling.

Expression of PIWIL2 in oral cancer and leukoplakia: Prognostic implications and insights from tumors.

PIWIL2 is a human Argonaute protein, which is guided by small RNAs to its targets, plays a role in germ cell maintenance and has been proposed to be expressed in precancerous stem cells and tumor stem cells. However, the significance of PIWIL2 expression in oral cancer and precancerous lesions has not been investigated. In this study, we analyzed the expression of the stem cell protein PIWIL2 in oral squamous-cell carcinoma (OSCC) and in premalignant oral leukoplakia (OL) with predominant expression in malignant and premalignant tissues. In the evaluated patients, we found that PIWIL2 was associated significantly with OSCC prognosis and OL. Furthermore, PIWIL2 was found to be expressed in tumor epithelial cells and macrophages in the tumor microenvironment, which are not derived from enlarged lymph nodes. Cytological experiments confirmed that the human squamous cell carcinoma cell line SCC-25, can promote the PIWIL2 and Nanog level in THP-1 cells, which are extensively used to study the modulation of monocytes and macrophages. Our findings showed that PIWIL2 can predict effectively OSCC prognosis and OL with a high risk of OSCC development and substantiate the deduction that cancer stem(-like) cells in oral cancer have the ability to reconstitute the heterogeneity of the bulk tumor and contribute to poor outcome and immunosuppression.

Leptin treatment of in vitro cultured embryos increases outgrowth rate of inner cell mass during embryonic stem cell derivation.

Leptin, a metabolic hormone, regulates the reproductive functions responding to both nutritional and body conditions. Embryonic stem cells play important roles in reproductive technology, but their derivation can be challenging. In this study, we evaluated the derivation rates of mouse embryonic stem cell (mESC) line from blastocysts developing in embryo culture media supplemented with different leptin concentrations. The results showed that addition of leptin into the embryo culture medium supported the in vitro development of mouse embryo. The mESC line derivation rates for media treated with 0, 10, 50, and 100 ng/ml of leptin were 61.24 % (54/88), 84.96 % (42/50), 81.79 % (61/76), and 85.78 % (56/67), respectively. In addition, leptin treatment of blastocysts upregulated the expression levels of the trophectoderm marker Cdx2, whereas inner cell mass markers Oct-4 and Nanog were not affected. mESC lines derived after leptin treatment demonstrated hallmarks of pluripotency, such as alkaline phosphatase activity, expression of, OCT4, NANOG, and SSEA1, as well as the ability to form embryoid bodies and well-differentiated teratomas. In conclusion, leptin has a positive effect on the derivation rate of mouse embryonic stem cell lines which may be, in part, due to its effects on the development of the trophectoderm cell lineage in the embryo.

ALDH1 expression correlates with an epithelial-like phenotype and favorable prognosis in lung adenocarcinoma: a study based on immunohistochemistry and mRNA expression data.

PURPOSE: Cancer stem cells (CSC) and epithelial-mesenchymal transition (EMT) pathways are crucial for cancer progression. However, synergistic interactions between CSC and EMT are not clear in non-small cell lung cancer (NSCLC). The objective of this study was to investigate CSC markers such as CD44, NANOG, and ALDH1 expression and its correlation with EMT markers in NSCLC patients. Its association with survival was also determined. METHODS: CD44, NANOG, and ALDH1 protein expression was evaluated in 267 resected NSCLC and its correlation with e-cadherin, ß-catenin, p120 catenin, vimentin, SNAIL, and TWIST expressions was determined based on immunohistochemical and mRNA expression data from The Cancer Genome Atlas (TCGA) database. Survival analyses also were performed based on immunohistochemistry and mRNA expression data from Gene Expression Omnibus dataset. RESULTS: ALDH1 expression in lung adenocarcinoma was positively correlated with the epithelial-like phenotype, low vimentin and low TWIST in immunohistochemical and mRNA expression data. NANOG and ALDH1 expressions measured by immunohistochemical and mRNA expression profiling data of adenocarcinomas were associated with a favorable prognosis. ALDH1 was an independent favorable prognostic marker for overall survival or recurrence-free survival in adenocarcinoma (P = 0.026 and P = 0.033, respectively). The epithelial-like phenotype expressing P120-catenin and beta-catenin was associated with a favorable prognosis; however, the TWIST-expressing mesenchymal-like phenotype was correlated with an unfavorable prognosis. CONCLUSIONS: NANOG and ALDH1 protein or mRNA expression showed improved prognosis in adenocarcinoma alone. ALDH1 expression correlated with an epithelial-like phenotype.

Human amniotic fluid stem cells (hAFSCs) expressing p21 and cyclin D1 genes retain excellent viability after freezing with (dimethyl sulfoxide) DMSO.

Human amniotic fluid stem cells (hAFSCs) have features intermediate between embryonic and adult SCs, can differentiate into lineages of all three germ layers, and do not develop into tumors in vivo. Moreover, hAFSCs can be easily obtained in routine procedures and there is no ethical or legal limitations regarding their use for clinical and experimental applications. The aim of this study was to assess the effect of slow freezing/thawing and two different concentrations of DMSO (10% DMSO + 90% fetal bovine serum [FBS] and 5% DMSO + 95% FBS) on the survival of hAFSCs. hAFSCs were obtained from 5 pregnant women during amniocentesis at 16-22 weeks of gestation. The expression of pluripotency markers (Octamer-binding transcription factor 4 [Oct4] and NANOG) by reverse transcription polymerase chain reaction and cell surface markers (cluster of differentiation [CD31], CD44, CD45, and CD90) by flow cytometry was analyzed before and after the slow-freezing. Cell viability was assessed by trypan blue exclusion or MTT assay. Quantitative mRNA expression of Oct4, NANOG, cyclin D1 and p21 was determined by real-time PCR before and after the slow-freezing. Pluripotency of hAFSCs was confirmed by NANOG and POU5F1 (Oct4) gene expression before and after slow-freezing. All hAFSC cultures were positive for CD44 and CD90. A higher viability of hAFSCs was observed after freezing with 90% FBS + 10% DMSO. There was increased expression of NANOG and decreased expression of POU5F1 gene after freezing, compared to control cells (before freezing). DMSO and the process of freezing did not significantly change the expression of p21 and cyclin D1 genes in hAFSCs. Overall, our results indicate the applicability of slow-freezing and DMSO in cryopreservation of SCs.

IL-33 guides osteogenesis and increases proliferation and pluripotency marker expression in dental stem cells.

OBJECTIVES: Soluble IL-33 (interleukin (IL)-1-like cytokine) acts as endogenous alarm signal (alarmin). Since alarmins, besides activating immune system, act to restore tissue homeostasis, we investigated whether IL-33 exerts beneficial effects on oral stem cell pull. MATERIALS AND METHODS: Clonogenicity, proliferation, differentiation and senescence of stem cells derived from human periodontal ligament (PDLSCs) and dental pulp (DPSCs) were determined after in vitro exposure to IL-33. Cellular changes were detected by flow cytometry, Western blot, immunocytochemistry and semiquantitative RT-PCR. RESULTS: IL-33 stimulated proliferation, clonogenicity and expression of pluripotency markers, OCT-4, SOX-2 and NANOG, but it inhibited ALP activity and mineralization in both PDLSCs and DPSCs. Higher Ki67 expression and reduced ß-galactosidase activity in IL-33-treated cells were demonstrated, whereas these trends were more conspicuous in osteogenic medium. However, after 7-day IL-33 pretreatment, differentiation capacity of IL-33-pretreated cells was retained, and increased ALP activity was observed in both cell types. Results showed that IL-33 regulates NF-κB and ß-catenin signalling, indicating the association of these molecules with changes observed in IL-33-treated PDLSCs and DPSCs, particularly their proliferation, pluripotency-associated marker expression and osteogenesis. CONCLUSIONS: IL-33 treatment impairs osteogenesis of PDLSCs and DPSCs, while increases their clonogenicity, proliferation and pluripotency marker expression. After exposure to IL-33, osteogenic capacity of cells stayed intact. NF-κB and ß-catenin are implicated in the effects achieved by IL-33 in PDLSCs and DPSCs.

Immunoregulatory protein B7-H3 regulates cancer stem cell enrichment and drug resistance through MVP-mediated MEK activation.

B7-H3 is a tumor-promoting glycoprotein that is expressed at low levels in most normal tissues, but is overexpressed in various human cancers which is associated with disease progression and poor patient outcome. Although numerous publications have reported the correlation between B7-H3 and cancer progression in many types of cancers, mechanistic studies on how B7-H3 regulates cancer malignancy are rare, and the mechanisms underlying the role of B7-H3 in drug resistance are almost unknown. Here we report a novel finding that upregulation of B7-H3 increases the breast cancer stem cell population and promotes cancer development. Depletion of B7-H3 in breast cancer significantly inhibits the cancer stem cells. By immunoprecipitation and mass spectrometry, we found that B7-H3 is associated with the major vault protein (MVP) and activates MEK through MVP-enhancing B-RAF and MEK interaction. B7-H3 expression increases stem cell population by binding to MVP which regulates the activation of the MAPK kinase pathway. Depletion of MVP blocks the activation of MEK induced by B7-H3 and dramatically inhibits B7-H3 induced stem cells. This study reports novel functions of B7-H3 in regulating breast cancer stem cell enrichment. The novel mechanism for B7-H3-induced stem cell propagation by regulating MVP/MEK signaling axis independent of the classic Ras pathway may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

Partial regeneration of uterine horns in rats through adipose-derived stem cell sheets.

Severe uterine damage and infection lead to intrauterine adhesions, which result in hypomenorrhea, amenorrhea and infertility. Cell sheet engineering has shown great promise in clinical applications. Adipose-derived stem cells (ADSCs) are emerging as an alternative source of stem cells for cell-based therapies. In the present study, we investigated the feasibility of applying ADSCs as seed cells to form scaffold-free cell sheet. Data showed that ADSC sheets expressed higher levels of FGF, Col I, TGFß, and VEGF than ADSCs in suspension, while increased expression of this gene set was associated with stemness, including Nanog, Oct4, and Sox2. We then investigated the therapeutic effects of 3D ADSCs sheet on regeneration in a rat model. We found that ADSCs were mainly detected in the basal layer of the regenerating endometrium in the cell sheet group at 21 days after transplantation. Additionally, some ADSCs differentiated into stromal-like cells. Moreover, ADSC sheets transplanted into partially excised uteri promoted regeneration of the endometrium cells, muscle cells and stimulated angiogenesis, and also resulted in better pregnancy outcomes. Therefore, ADSC sheet therapy shows considerable promise as a new treatment for severe uterine damage.

Leucine-Rich Repeat Neuronal Protein 1 Regulates Differentiation of Embryonic Stem Cells by Post-Translational Modifications of Pluripotency Factors.

Stem cell surface markers may facilitate a better understanding of stem cell biology through molecular function studies or serve as tools to monitor the differentiation status and behavior of stem cells in culture or tissue. Thus, it is important to identify additional novel stem cell markers. We used glycoproteomics to discover surface glycoproteins on human embryonic stem cells (hESCs) that may be useful stem cell markers. We found that a surface glycoprotein, leucine-rich repeat neuronal protein 1 (LRRN1), is expressed abundantly on the surface of hESCs before differentiation into embryoid bodies (EBs). Silencing of LRRN1 with short hairpin RNA (shLRRN1) in hESCs resulted in decreased capacity of self-renewal, and skewed differentiation toward endoderm/mesoderm lineages in vitro and in vivo. Meanwhile, the protein expression levels of the pluripotency factors OCT4, NANOG, and SOX2 were reduced. Interestingly, the mRNA levels of these pluripotency factors were not affected in LRRN1 silenced cells, but protein half-lives were substantially shortened. Furthermore, we found LRRN1 silencing led to nuclear export and proteasomal degradation of all three pluripotency factors. In addition, the effects on nuclear export were mediated by AKT phosphorylation. These results suggest that LRRN1 plays an important role in maintaining the protein stability of pluripotency factors through AKT phosphorylation, thus maintaining hESC self-renewal capacity and pluripotency. Overall, we found that LRRN1 contributes to pluripotency of hESC by preventing translocation of OCT4, NANOG, and SOX2 from nucleus to cytoplasm, thereby lessening their post-translational modification and degradation. Stem Cells 2018;36:1514-1524.

ERK inhibition promotes neuroectodermal precursor commitment by blocking self-renewal and primitive streak formation of the epiblast.

BACKGROUND: Pluripotent stem cells hold great promise for regenerative medicine. However, before clinical application, reproducible protocols for pluripotent stem cell differentiation should be established. Extracellular signal-regulated protein kinase (ERK) signaling plays a central role for the self-renewal of epiblast stem cells (EpiSCs), but its role for subsequent germ layer differentiation is still ambiguous. We proposed that ERK could modulate differentiation of the epiblast. METHODS: PD0325901 was used to inhibit ERK activation during the differentiation of embryonic stem cells and EpiSCs. Immunofluorescence, western blot analysis, real-time PCR and flow cytometry were used to detect germ layer markers and pathway activation. RESULTS: We demonstrate that the ERK phosphorylation level is lower in neuroectoderm of mouse E7.5 embryos than that in the primitive streak. ERK inhibition results in neural lineage commitment of epiblast. Mechanistically, PD0325901 abrogates the expression of primitive streak markers by ß-catenin retention in the cytoplasm, and inhibits the expression of OCT4 and NANOG during EpiSC differentiation. Thus, EpiSCs differentiate into neuroectodermal lineage efficiently under PD0325901 treatment. These results suggest that neuroectoderm differentiation does not require extrinsic signals, supporting the default differentiation of neural lineage. CONCLUSIONS: We report that a single ERK inhibitor, PD0325901, can specify epiblasts and EpiSCs into neural-like cells, providing an efficient strategy for neural differentiation.

The Prognostic Value of Nanog Overexpression in Lung Cancer: A Meta-Analysis.

BACKGROUND: Recent several studies have showed that the nanog overexpression leads to poor prognosis in some kinds of cancer including hepatocellular carcinoma and gastrointestinal luminal cancer. However, the correlations between prognosis and clinic-pathological features and nanog overexpression in lung cancer are still not well-known. Thus, we performed a meta-analysis to evaluate the role of nanog in lung cancer. METHODS: An electronic retrieval for related studies was conducted in PubMed, Cochrane Library, Web of Science, EMBASE databases, Chinese CNKI, and the Chinese Wan Fang database up to May 2018. The relationships between nanog overexpression and overall survival (OS) and disease-free survival (DFS) as well as clinic-pathological features in lung cancer were investigated. Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by STATA12. RESULTS: 11 studies containing 1422 patients were identified in our meta-analysis. The overexpression of nanog showed decreased OS (HR = 1.83, 95% CI = 1.49-2.25, P ≤ 0.001) and DFS (HR = 1.86, 95% CI = 1.2-2.9, P = 0.006). Moreover, overexpression of nanog was significantly related to differentiation (OR = 4.17, 95% CI = 2.17-6.43, P ≤ 0.001), lymph node metastasis (OR = 1.76, 95% CI = 1.06-2.91, P = 0.028) and tumor size (OR = 1.93, 95% CI = 1.17-3.20, P = 0.010), and no correlation with T stage, TNM, stage, and gender. CONCLUSIONS: Our results suggested that nanog overexpression, a hazard factor of differentiation, lymph node metastasis, and tumor size, may predicate decreased OS and DFS for lung cancer.

The primary role of zebrafish nanog is in extra-embryonic tissue.

The role of the zebrafish transcription factor Nanog has been controversial. It has been suggested that Nanog is primarily required for the proper formation of the extra-embryonic yolk syncytial layer (YSL) and only indirectly regulates gene expression in embryonic cells. In an alternative scenario, Nanog has been proposed to directly regulate transcription in embryonic cells during zygotic genome activation. To clarify the roles of Nanog, we performed a detailed analysis of zebrafish nanog mutants. Whereas zygotic nanog mutants survive to adulthood, maternal-zygotic (MZnanog) and maternal mutants exhibit developmental arrest at the blastula stage. In the absence of Nanog, YSL formation and epiboly are abnormal, embryonic tissue detaches from the yolk, and the expression of dozens of YSL and embryonic genes is reduced. Epiboly defects can be rescued by generating chimeric embryos of MZnanog embryonic tissue with wild-type vegetal tissue that includes the YSL and yolk cell. Notably, cells lacking Nanog readily respond to Nodal signals and when transplanted into wild-type hosts proliferate and contribute to embryonic tissues and adult organs from all germ layers. These results indicate that zebrafish Nanog is necessary for proper YSL development but is not directly required for embryonic cell differentiation.

Morphine delays neural stem cells differentiation by facilitating Nestin overexpression.

BACKGROUND: Morphine is used as an analgesic although it causes important secondary effects. These effects are triggered by several mechanisms leading to the dysregulation of gene expression. Here we aimed to study these alterations on neural stem cells (NSC) during CNS development. METHODS: AB strain and tg nestin:GFP zebrafish embryos, zebrafish primary neuron culture and mouse embryonic stem cells were used to assess the effect of morphine by qPCR, time lapse microscopy and western blot. ChIP-qPCR and bisulfite conversion assay were performed to determine the changes exerted by morphine in a Nestin candidate enhancer. RESULTS: Morphine increases GFP in nestin:GFP embryos and overexpresses the NSC marker Nestin. Morphine also exerts a hyperacetylation effect on H3K27 and decreases DNA methylation within a region located 18 Kb upstream nestin transcription starting site. Here, a binding site for the transcription factor complex Sox2/Oct4/Nanog was predicted. These factors are also upregulated by morphine. Besides, morphine increases the histone acetyl transferase p300. The inhibition of p300 activity decreases Nestin. CONCLUSIONS: Morphine facilitates Nestin increase by several mechanisms which include hyperacetylation of H3K27, decreased DNA methylation and the overexpression of the transcription factors sox2, oct4 and nanog. It has also been demonstrated that nestin levels depend on p300 activity. The facilitated Nestin expression delays the normal differentiation of neural stem cells. GENERAL SIGNIFICANCE: The present work provides novel evidence of the effects induced by morphine in the normal differentiation of NSCs, altering Nestin through changes on p300, H3K27ac, DNA methylation and Oct4, Sox2, and Nanog.

Immunohistochemical expression of NANOG in urothelial carcinoma of the bladder.

Urothelial carcinoma is a common malignant neoplasm that has a poor prognosis and a high frequency of recurrence and metastasis. Constant disease surveillance with periodic and long term cystoscopy examination is necessary for management of the disease. However, the monitoring and therapy regimen is expensive, incurring a massive burden to patients and the government. Therefore, the development of specific biomarkers for urothelial carcinoma at an early stage and recurrence detection becomes a priority. Homeobox genes are a family of genes that are involved in tumourigenesis. They might be potential prognostic markers for urothelial carcinoma. The study investigated the expression pattern of NANOG which is one of a homeobox gene in different stages and grades of urothelial carcinoma. NANOG expressions were also correlated with patient demographic factors and clinicopathological parameters. The expression of NANOG in 100 formalin-fixed paraffin-embedded urothelial carcinoma tissues was determined by immunohistochemistry. Immunohistochemistry showed positive expression of NANOG in all specimens with detection in the cytoplasm, nuclei and the nuclear membrane of the cancer cells. The immunohistochemical expression of NANOG increased across stages and grades of the tumour. The expression of NANOG was not significantly associated with demographic factors; gender (p = 0.376), race (p = 0.718) and age (p = 0.058) as well as with most of the clinicopathological parameters; pathological stage (p = 0.144), grade (p = 0.625), lymph node involvement (p = 0.174) and distant metastasis (p = 0.228). However, NANOG expression showed significant correlation with tumour invasion (p = 0.019). We concluded that NANOG might be a potential biomarker for early diagnosis of urothelial carcinoma of the bladder.

GLI2 induces PDGFRB expression and modulates cancer stem cell properties of gastric cancer.

OBJECTIVE: In this study, we aimed to investigate the downstream effector of GLI2 in gastric cancer (GC) and their regulative effect on cancer stem cell (CSC) properties of GC. MATERIALS AND METHODS: Bioinformatic data mining was performed in TCGA-Stomach Adenocarcinoma (STAD), as well as in Kaplan-Meier plotter. Moderate-differentiated GC cell line SGC-7901 and poor-differentiated GC cell line MKN-45 were used as in-vitro model to investigate the regulative effect of GLI2 on PDGFRB expression. MKN-45 cells were further used to explore the effect of GLI2 shRNA or PDGFRB shRNA on CSC properties of the cells. RESULTS: Bioinformatic results showed that GLI2 is usually upregulated in GC tissues than in normal tissues, and high GLI2 expression is associated with unfavorable first progression free survival (PFS) and also worse overall survival (OS) in patients with GC. PDGFRB is co-upregulated with GLI2 in GC and its promoter region contains a putative GLI2 binding site. The results of dual luciferase assay confirmed this binding site. Enforced GLI2 expression elevated PDGFRB expression at both mRNA and protein level. GLI2 or PDGFRB knockdown showed similar effect on reducing spheroid colony formation and on reducing the expression of CSC related genes, including CD44, Nanog, and Oct4 in MKN-45 cells. CONCLUSIONS: High GLI2 or PDGFRB expression is associated with unfavorable survival in GC patients. GLI2 can induce PDGFRB expression in GC cells via directly binding to its promoter. In addition, the GLI2-PDGFRB axis might be an important signaling pathway modulating CSC properties of GC cells.

Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells.

Autophagy is required for benign hepatic tumors to progress into malignant hepatocellular carcinoma. However, the mechanism is unclear. Here, we report that mitophagy, the selective removal of mitochondria by autophagy, positively regulates hepatic cancer stem cells (CSCs) by suppressing the tumor suppressor p53. When mitophagy is enhanced, p53 co-localizes with mitochondria and is removed by a mitophagy-dependent manner. However, when mitophagy is inhibited, p53 is phosphorylated at serine-392 by PINK1, a kinase associated with mitophagy, on mitochondria and translocated into the nucleus, where it binds to the NANOG promoter to prevent OCT4 and SOX2 transcription factors from activating the expression of NANOG, a transcription factor critical for maintaining the stemness and the self-renewal ability of CSCs, resulting in the reduction of hepatic CSC populations. These results demonstrate that mitophagy controls the activities of p53 to maintain hepatic CSCs and provide an explanation as to why autophagy is required to promote hepatocarcinogenesis.