Nanog Transcription Research Articles

Targeting cancer stem cells: emerging role of Nanog transcription factor

The involvement of stemness factors in cancer initiation and progression has drawn much attention recently, especially after the finding that introducing four stemness factors in somatic cells is able to reprogram the cells back to an embryonic stem cell-like state. Following accumulating data revealing abnormal elevated expression levels of key stemness factors, like Nanog, Oct4, and Sox2, in several types of cancer stem cells; the importance and therapeutic potential of targeting these stemness regulators in cancers has turned to research focus. Nanog determines cell fate in both embryonic and cancer stem cells; activating Nanog at an inappropriate time would result in cancer stem cells rather than normal pluripotent stem cells or differentiated somatic cells. Upregulated Nanog is correlated with poor survival outcome of patients with various types of cancer. The discoveries of downstream regulatory pathways directly or indirectly mediated by Nanog indicate that Nanog regulates several aspects of cancer development such as tumor cell proliferation, self-renewal, motility, epithelial-mesenchymal transition, immune evasion, and drug-resistance, which are all defined features for cancer stem cells. The current review paper illustrates the central role of Nanog in the regulatory networks of cancer malignant development and stemness acquirement, as well as in the communication between cancer cells and the surrounding stroma. Though a more defined model is needed to test the therapeutic efficacy of targeting Nanog as a cancer treatment method, current animal experiments using siNanog or shNanog have shown the promising therapeutic potential of Nanog targeting in several types of cancer.

Distinct Allelic Patterns of Nanog Expression Impart Embryonic Stem Cell Population Heterogeneity

Nanog is a principal pluripotency regulator exhibiting a disperse distribution within stem cell populations in vivo and in vitro. Increasing evidence points to a functional role of Nanog heterogeneity on stem cell fate decisions. Allelic control of Nanog gene expression was reported recently in mouse embryonic stem cells. To better understand how this mode of regulation influences the observed heterogeneity of NANOG in stem cell populations, we assembled a multiscale stochastic population balance equation framework. In addition to allelic control, gene expression noise and random partitioning at cell division were considered. As a result of allelic Nanog expression, the distribution of Nanog exhibited three distinct states but when combined with transcriptional noise the profile became bimodal. Regardless of their allelic expression pattern, initially uniform populations of stem cells gave rise to the same Nanog heterogeneity within ten cell cycles. Depletion of NANOG content in cells switching off both gene alleles was slower than the accumulation of intracellular NANOG after cells turned on at least one of their Nanog gene copies pointing to Nanog state-dependent dynamics. Allelic transcription of Nanog also raises issues regarding the use of stem cell lines with reporter genes knocked in a single allelic locus. Indeed, significant divergence was observed in the reporter and native protein profiles depending on the difference in their half-lives and insertion of the reporter gene in one or both alleles. In stem cell populations with restricted Nanog expression, allelic regulation facilitates the maintenance of fractions of self-renewing cells with sufficient Nanog content to prevent aberrant loss of pluripotency. Our findings underline the role of allelic control of Nanog expression as a prime determinant of stem cell population heterogeneity and warrant further investigation in the contexts of stem cell specification and cell reprogramming.

Connective Tissue Growth Factor Activates Pluripotency Genes and Mesenchymal–Epithelial Transition in Head and Neck Cancer Cells

The epithelial-mesenchymal transition (EMT) is a key mechanism in both embryonic development and cancer metastasis. The EMT introduces stem-like properties to cancer cells. However, during somatic cell reprogramming, mesenchymal-epithelial transition (MET), the reverse process of EMT, is a crucial step toward pluripotency. Connective tissue growth factor (CTGF) is a multifunctional secreted protein that acts as either an oncoprotein or a tumor suppressor among different cancers. Here, we show that in head and neck squamous cell carcinoma (HNSCC), CTGF promotes the MET and reduces invasiveness. Moreover, we found that CTGF enhances the stem-like properties of HNSCC cells and increases the expression of multiple pluripotency genes. Mechanistic studies showed that CTGF induces c-Jun expression through αvβ3 integrin and that c-Jun directly activates the transcription of the pluripotency genes NANOG, SOX2, and POU5F1. Knockdown of CTGF in TW2.6 cells was shown to reduce tumor formation and attenuate E-cadherin expression in xenotransplanted tumors. In HNSCC patient samples, CTGF expression was positively correlated with the levels of CDH1, NANOG, SOX2, and POU5F1. Coexpression of CTGF and the pluripotency genes was found to be associated with a worse prognosis. These findings are valuable in elucidating the interplay between epithelial plasticity and stem-like properties during cancer progression and provide useful information for developing a novel classification system and therapeutic strategies for HNSCC.

Acetylated histone H3K56 interacts with Oct4 to promote mouse embryonic stem cell pluripotency

The presence of acetylated histone H3K56 (H3K56ac) in human ES cells (ESCs) correlates positively with the binding of Nanog, Sox2, and Oct4 (NSO) transcription factors at their target gene promoters. However, the function of H3K56ac there has been unclear. We now report that Oct4 interacts with H3K56ac in mouse ESC nuclear extracts and that perturbing H3K56 acetylation decreases Oct4-H3 binding. This interaction is likely to be direct because it can be recapitulated in vitro in an H3K56ac-dependent manner and is functionally important because H3K56ac combines with NSO factors in chromatin immunoprecipitation sequencing to mark the regions associated with pluripotency better than NSO alone. Moreover, reducing H3K56ac by short hairpin Asf1a decreases expression of pluripotency-related markers and increases expression of differentiation-related ones. Therefore, our data suggest that H3K56ac plays a central role in binding to Oct4 to promote the pluripotency of ESCs.

Effects of DNMT1 and HDAC Inhibitors on Gene-Specific Methylation Reprogramming during Porcine Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) in mammalian cloning currently remains inefficient. Incomplete or erroneous epigenetic reprogramming of specialized donor somatic nuclear and resulting aberrant gene expression during development of cloned embryos is commonly believed as the main reason that causes the low efficiency of SCNT. Use of small molecular reprogramming modifiers to assist the somatic nucleus to mimic naturally occurring DNA methylation and chromatin remodeling in nucleus of fertilization-derived zygotes, has been widely attempted to improve cloning efficiency. However, impacts of these small modifiers on gene-specific methylation dynamics and their potential effects on methylation of imprinted gene have rarely been traced. Here, we attempted two relatively novel DNMT1 inhibitor (DNMTi) and histone deacetylase inhibitor (HDACi), scriptaid and RG108, and demonstrated their effects on dynamics of gene-specific DNA methylation and transcription of porcine SCNT embryos. We found that scriptaid and RG108 had synergetic effects on rescuing the disrupted methylation imprint of H19 during SCNT at least partially by repression over-expressed MBD3 in eight-cell cloned embryos. Furthermore, we firstly identified a differential methylation regions (DMRs) at 5′ flanking regions of XIST gene and found that scriptaid alone and its combination with RG108 modify the dynamics of both transcription and DNA methylation levels in cloned embryos, by different manners. Additionally, we found that scriptaid alone and its combination with RG108 can significantly promote the transcription of NANOG in cloned embryos and enhance their pre-implantation developmental capacity. Our results would contribute to uncovering the epigenetic reprogramming mechanisms underlying the effects of assisted small molecules on improvement of mammalian cloning efficiency.

RNA-binding protein Rbm47 binds to Nanog in mouse embryonic stem cells

Embryonic stem cells (ES cells) are pluripotent cells capable for self-renewal and to differentiate to all cell types. Finding the molecular mechanisms responsible for these unique characteristics of ES cells is important. RNA-binding proteins play important roles in post-transcriptional gene regulation by binding to specific mRNA targets. In this study, we investigated the targets of RNA-binding protein Rbm47 in mouse ES cells. Overexpression of HA epitope-tagged Rbm47 in mouse ES cells followed by RNA-binding protein immunoprecipitation, and then RT-PCR analysis of co-immunoprecipitated RNA showed that Rbm47 binds to Nanog transcript in mouse ES cells and doesn't bind to Sox2 and Oct4 transcripts in these cells. This finding can give rise to reveal molecular mechanisms underlying pluripotency and stemness of ES cells and will be necessary for efficient application of these cells in regenerative medicine and tissue engineering.

Zic3 Enhances the Generation of Mouse Induced Pluripotent Stem Cells

Zinc finger protein of the cerebellum (Zic)3, a member of Gli family of transcription factors (TFs), is essential for maintaining pluripotency of embryonic stem cells (ESCs) and has been reported to activate TF Nanog in an Oct4/Sox2-independent manner. Previously, we showed that Zic3 (Z), in combination with the Yamanka factors OCT4, SOX2, and KLF4 (OSK), induces neural progenitor-like cells from human fibroblasts. However, a similar combination of TFs (OSKZ) transduced in mouse embryonic fibroblasts resulted in enhanced induced pluripotent stem cells (iPSCs) formation compared with OSK alone, but not neuroprogenitors. OSKZ-derived iPSCs are indistinguishable from mESCs in colony morphology, expression of alkaline phosphatase and pluripotency genes, and embryoid body and teratoma formation. Zic3 activates the transcription of Nanog, a key pluripotency regulator, as evidenced by a luciferase promoter assay. During the course of iPSC derivation, Zic3-mediated enhanced expression of Nanog and Tbx3, gene known to enhance iPSCs derivation, is observed. Not only does Zic3 enhance the reprogramming efficiency, but also reactivation of the endogenous Zic3 protein is essential for the generation of iPSCs, as knockdown of Zic3 during the iPSC generation with OSKM significantly reduced the number of colonies. Together, our result uncovers an important role of Zic3 in generating mouse iPSCs.

Inhibition of MAP2K and GSK3 Signaling Promotes Bovine Blastocyst Development and Epiblast-Associated Expression of Pluripotency Factors1

Cells in the mammalian blastocyst segregate into three distinct lineages, namely, trophoblast, hypoblast, and epiblast. During development, these will form extraembryonic and embryonic tissues, respectively. In mouse, only epiblast cells can be directly converted into cultured pluripotent embryonic stem cells, capable of forming all adult cell types. This conversion is promoted by the double inhibition (i.e., 2i) of mitogen-activated protein kinase kinase (Map2k), antagonizing Fgf signaling, and of glycogen synthase kinase 3 (Gsk3), stimulating the Wnt pathway. We investigated the effect of 2i treatment on lineage segregation and pluripotency-related gene expression in bovine blastocysts. In vitro fertilized (IVF) embryos were cultured in the presence of dimethyl sulfoxide or inhibitors of MAP2K (0.4 μM PD0325901) and GSK3 (3 μM CHIR99021) from the zygote (Day 1) stage. Compared to vehicle controls, 2i conditions increased the abundance of cumulus cells in bovine IVF cultures, which compromised blastocyst formation. Following cumulus removal, 2i accelerated blastocyst development and increased inner cell mass (ICM) and trophoblast cell numbers by 30% and 27%, respectively. These developmental and morphological changes were accompanied by alterations in gene expression. Signal inhibition increased transcription of putative epiblast markers NANOG and SOX2 while repressing putative hypoblast marker GATA4. Using microsurgical blastocyst dissection, we found that the increase in NANOG and SOX2 levels was specific to the ICM and not due to ectopic expression in the trophoblast. Expression of other pluripotency-related (POU5F1, KLF4, DPPA3) or trophoblast-enriched (CDX2) genes was not affected. In summary, 2i conditions reprogrammed the transcriptional profile of bovine ICM but not trophoblast cells. By shifting the balance from hypoblast- to epiblast-associated gene expression, 2i culture may prime bovine epiblast for subsequent derivation of pluripotent stem cell cultures.

Autochthonous Mouse Melanoma and Mammary Tumors do not Express the Pluripotency Genes Oct4 and Nanog

The homeodomain transcription factors Oct4 and Nanog maintain pluripotency and self-renewal in embryonic stem cells. In somatic cells, inappropriate expression of these genes has been associated with loss of differentiation, malignant transformation, and the acquisition of cancer stem cell-like properties. As cancer stem cells have been suggested to underlie the growth and malignancy of tumors, Oct4 and Nanog may represent therapeutic targets. Their expression could also act as a marker of the cancer stem cell population, permitting its isolation and characterisation. Nevertheless, the existence of multiple pseudogenes and isoforms of these genes has complicated the interpretation of the data that supports a role for Oct4 and Nanog in the cancer context. Here we addressed this issue using knockin mice in which IRES elements are used to allow GFP expression under the control of the endogenous Oct4 or Nanog promoters, while maintaining correct expression of the Oct4 or Nanog gene. These mice were crossed with MT/ret mice that develop melanomas, and with MMTV-PyMT mice and MMTV-Neu mice that develop mammary adenocarcinomas. We analysed the tumors that developed in these compound mice for GFP expression. In this way we could assess transcription of Oct4 and Nanog in autochthonous cancers without the complication of factors such as pseudogene expression, alternative splicing and antibody specificity. Both the Oct4 and Nanog knockin tumor-bearing mice expressed GFP in blastocysts and testes as expected. However, we could find no evidence for expression of the GFP reporter above background levels in tumors using FACS, qPCR and immunohistochemistry. Furthermore, cultivation of Oct4GFP and NanogGFP MMTV-PyMT tumor cells either adherently or as spheroids had no effect on the expression of the GFP reporter. Together these data suggest that Oct4 and Nanog are not expressed in tumor cells that arise in the autochthonous cancer models studied here.

Nanog Is an Essential Factor for Induction of Pluripotency in Somatic Cells from Endangered Felids

Nanog has an important role in pluripotency induction in bovines and snow leopards. To examine whether it was required for wild felids globally, we examined the induction of pluripotency in felids from Asia (Bengal tiger, Panthera tigris), Africa (serval, Leptailurus serval), and the Americas (jaguar, Panthera onca). Dermal fibroblasts were transduced with genes encoding the human transcription factors OCT4, SOX2, KLF4, and cMYC with or without NANOG. Both four- and five-factor induction resulted in colony formation at day 3 in all three species tested; however, we were not able to maintain colonies that were generated without NANOG beyond passage (P) 7. Five-factor induced pluripotent stem cell (iPSC) colonies from wild cats were expanded in vitro on feeder layers and were positive for alkaline phosphatase and protein expression of OCT-4, NANOG, and stage-specific embryonic antigen-4 at P4 and P14. Reverse-transcription polymerase chain reaction confirmed that all five human transgenes were transcribed at P4; however, OCT4, SOX2, and NANOG transgenes were silenced by P14. Endogenous OCT4 and NANOG transcripts were detected at P4 and P14 in all cell lines confirming successful reprogramming. At P14, the iPSCs from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers. This study describes an effective method for inducing pluripotency in three endangered wild cats from across the globe and confirms Nanog as an essential factor in the reprogramming event. Efficient production of iPSC from endangered felids creates a unique opportunity for species preservation through gamete production, nuclear transfer, embryo complementation, and future novel technologies.

Global DNA Hypomethylation Prevents Consolidation of Differentiation Programs and Allows Reversion to the Embryonic Stem Cell State

DNA methylation patterns change dynamically during mammalian development and lineage specification, yet scarce information is available about how DNA methylation affects gene expression profiles upon differentiation. Here we determine genome-wide transcription profiles during undirected differentiation of severely hypomethylated (Dnmt1−/−) embryonic stem cells (ESCs) as well as ESCs completely devoid of DNA methylation (Dnmt1−/−;Dnmt3a−/−;Dnmt3b−/− or TKO) and assay their potential to transit in and out of the ESC state. We find that the expression of only few genes mainly associated with germ line function and the X chromosome is affected in undifferentiated TKO ESCs. Upon initial differentiation as embryoid bodies (EBs) wild type, Dnmt1−/− and TKO cells downregulate pluripotency associated genes and upregulate lineage specific genes, but their transcription profiles progressively diverge upon prolonged EB culture. While Oct4 protein levels are completely and homogeneously suppressed, transcription of Oct4 and Nanog is not completely silenced even at late stages in both Dnmt1−/− and TKO EBs. Despite late wild type and Dnmt1−/− EBs showing a much higher degree of concordant expression, after EB dissociation and replating under pluripotency promoting conditions both Dnmt1−/− and TKO cells, but not wild type cells rapidly revert to expression profiles typical of undifferentiated ESCs. Thus, while DNA methylation seems not to be critical for initial activation of differentiation programs, it is crucial for permanent restriction of developmental fate during differentiation.

Gata4 Blocks Somatic Cell Reprogramming By Directly Repressing Nanog

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by ectopic expression of the four factors Oct4, Klf4, Sox2, and Myc. Here, we investigated the role of Gata4 in the reprogramming process and present evidence for a negative role of this family of transcription factors in the induction of pluripotency. Coexpression of Gata4 with Oct4, Klf4, and Sox2 with or without Myc in mouse embryonic fibroblasts greatly impaired reprogramming and endogenous Nanog expression. The lack of Nanog upregulation was associated with a blockade in the transition from the initiation phase of reprogramming to the full pluripotent state characteristic of iPS cells. Addition of Nanog to the reprogramming cocktail blocked the deleterious effects observed with Gata4 expression. Downregulation of endogenous Gata4 by short hairpin RNAs during reprogramming both accelerated and increased the efficiency of the process and augmented the mRNA levels of endogenous Nanog. Using comparative genomics, we identified a consensus binding site for Gata factors in an evolutionary conserved region located 9 kb upstream of the Nanog gene. Using chromatin immunoprecipitation, gel retardation, and luciferase assays, we found that Gata4 bound to this region and inhibited Nanog transcription in mouse embryonic stem cells. Overall, our results describe for first time the negative effect of Gata4 in the reprogramming of somatic cells and highlight the role of Gata factors in the transcriptional networks that control cell lineage choices in the early embryo.

Targeting TCTP as a New Therapeutic Strategy in Castration-resistant Prostate Cancer

Heat shock protein 27 (Hsp27) is highly overexpressed in castration-resistant prostate cancer (CRPC) and an antisense inhibitor (OGX-427) is currently in phase II clinical trials. In order to understand mechanisms of action of Hsp27 and find new therapeutic targets specific of CRPC, we screened for Hsp27 client proteins. Here, we report that translationally controlled tumor protein (TCTP) is a new Hsp27 client protein involved in Hsp27 cytoprotection. We found that TCTP expression is absent or weak in normal prostate cells, moderately expressed in 18.5% of treatment naive PC, and becomes uniformly and strongly expressed in 75% of CRPC. To define TCTP function, we developed and worldwide patented a TCTP antisense oligonucleotide (ASO). Interestingly, we found that CRPC progression correlates with TCTP overexpression and loss of P53. TCTP knockdown restored P53 expression and function, suggesting that castration-sensitivity is directly linked to P53 expression. Collectively, these findings provide a new Hsp27 cytoprotection mechanism in CRPC, and preclinical proof-of-concept that combining ASO-mediated TCTP knockdown with castration and/or docetaxel therapy could serve as a novel strategy to treat CRPC, with no or little toxicity for normal prostate cells.

OCT4/SOX2-independent Nanog autorepression modulates heterogeneous Nanog gene expression in mouse ES cells.

NANOG, OCT4 and SOX2 form the core network of transcription factors supporting embryonic stem (ES) cell self-renewal. While OCT4 and SOX2 expression is relatively uniform, ES cells fluctuate between states of high NANOG expression possessing high self-renewal efficiency, and low NANOG expression exhibiting increased differentiation propensity. NANOG, OCT4 and SOX2 are currently considered to activate transcription of each of the three genes, an architecture that cannot readily account for NANOG heterogeneity. Here, we examine the architecture of the Nanog-centred network using inducible NANOG gain- and loss-of-function approaches. Rather than activating itself, Nanog activity is autorepressive and OCT4/SOX2-independent. Moreover, the influence of Nanog on Oct4 and Sox2 expression is minimal. Using Nanog:GFP reporters, we show that Nanog autorepression is a major regulator of Nanog transcription switching. We conclude that the architecture of the pluripotency gene regulatory network encodes the capacity to generate reversible states of Nanog transcription via a Nanog-centred autorepressive loop. Therefore, cellular variability in self-renewal efficiency is an emergent property of the pluripotency gene regulatory network.

Hyaluronan-CD44v3 Interaction with Oct4-Sox2-Nanog Promotes miR-302 Expression Leading to Self-renewal, Clonal Formation, and Cisplatin Resistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

Early embryonic lethality of mice with disrupted transcription cofactor PIMT/NCOA6IP/Tgs1 gene

PIMT (also known as PIPMT/NCOA6IP/Tgs1), first isolated as a transcription coactivator PRIP (NCOA6)-interacting 96-kDa protein with RNA-binding property, possesses RNA methyltransferase activity. As a transcription coactivator binding protein, PIMT enhances the nuclear receptor transcriptional activity and its methyltransferase property is involved in the formation of the 2,2,7-trimethylguanosine cap of non-coding small RNAs, but the in vivo functions of this gene have not been fully explored. To elucidate the biological functions, we used gene targeting to generate mice with a disrupted PIMT/Tgs1 gene. Disruption of PIMT gene results in early embryonic lethality due to impairment of development around the blastocyst and uterine implantation stages. We show that PIMT is expressed in all cells of the E3.5day blastocyst in the mouse. PIMT null mutation abolished PIMT expression in all cells of the blastocyst and caused a reduction in the expression of Oct4 and Nanog transcription factor proteins in the E3.5 blastocyst resulting in the near failure to form inner cell mass (ICM). With conditional deletion of PIMT gene, mouse embryonic fibroblasts (MEFs) exhibit defective wound healing in the scratch assay and a reduction in cell proliferation due to decreased G0/G1 transition and G2/M phase cell cycle arrest. We conclude that PIMT/NCOA6IP, which is expressed in all cells of the 3.5day stage blastocyst, is indispensable for early embryonic development.

E-cadherin and, in Its Absence, N-cadherin Promotes Nanog Expression in Mouse Embryonic Stem Cells via STAT3 Phosphorylation

We have recently shown that loss of E-cadherin in mouse embryonic stem cells (mESCs) results in significant alterations to both the transcriptome and hierarchy of pluripotency-associated signaling pathways. Here, we show that E-cadherin promotes kruppel-like factor 4 (Klf4) and Nanog transcript and protein expression in mESCs via STAT3 phosphorylation and that β-catenin, and its binding region in E-cadherin, is required for this function. To further investigate the role of E-cadherin in leukemia inhibitory factor (LIF)-dependent pluripotency, E-cadherin null (Ecad(-/-)) mESCs were cultured in LIF/bone morphogenetic protein supplemented medium. Under these conditions, Ecad(-/-) mESCs exhibited partial restoration of cell-cell contact and STAT3 phosphorylation and upregulated Klf4, Nanog, and N-cadherin transcripts and protein. Abrogation of N-cadherin using an inhibitory peptide caused loss of phospho STAT3, Klf4, and Nanog in these cells, demonstrating that N-cadherin supports LIF-dependent pluripotency in this context. We therefore identify a novel molecular mechanism linking E- and N-cadherin to the core circuitry of pluripotency in mESCs. This mechanism may explain the recently documented role of E-cadherin in efficient induced pluripotent stem cell reprogramming.

Fibroblast Growth Factor Receptor 2 Homodimerization Rapidly Reduces Transcription of the Pluripotency Gene Nanog without Dissociation of Activating Transcription Factors

Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.

Cytoplasmic NANOG-Positive Stromal Cells Promote Human Cervical Cancer Progression

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell gene NANOG, a divergent homeodomain transcription factor that is independent of leukemia inhibitory factor, has been reported to be expressed in germ cells and in several tumor types. However, the short-term expression and role of NANOG in cervical cancer remain unclear. In the present study, we demonstrate that NANOG exhibits cellular shuttling behavior and increasing stromal distribution during the progression of cervical cancer. Our molecular data using RT-PCR and restriction enzyme digestion show that NANOG is mainly transcribed from the NANOG gene in cervical cancer. In addition, IHC using confocal microscopy suggests that mesenchymal stem cells (MSCs) are one type of cytoplasmic NANOG-positive cells in cervical cancer stroma. Co-culture of cervical cancer-derived MSCs with SiHa cells showed increased proliferation characteristics in vitro and enhanced tumor growth in vivo. Our results show, for the first time to our knowledge, that MSCs are a source of cytoplasmic NANOG expression in the cervical cancer stroma and that they participate in the progression of cervical cancer both in vitro and in vivo. Our study provides evidence that NANOG is a cervical cancer progression marker and also serves as a starting point for a more extensive exploration of the cellular translocation of NANOG and the multifunctionality of the stromal microenvironment.

Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling.