Nanog Promoter Activity Research Articles

Epithelium-specific ETS transcription factor-1 regulates NANOG expression and inhibits NANOG-induced proliferation of human embryonic carcinoma cells

The epithelium-specific ETS transcription factor-1 (ESE-1) plays multiple roles in pathogenesis and normal development of epithelial tissues. NANOG, a key mediator of stem cell self-renewal and pluripotency, is also expressed in various cancers and pluripotent cells. In this study, we investigated how ESE-1 influences NANOG expression and NANOG-induced proliferation in human germ cell-derived embryonic carcinoma NCCIT cells. Endogenous ESE-1 expression in NCCIT cells significantly increased during differentiation, whereas NANOG expression decreased. In addition, NANOG expression was downregulated by exogenous overexpression of ESE-1, and increased by shRNA-mediated knockdown of ESE-1. NANOG transcriptional activity was reduced by dose-dependent ESE-1 overexpression and a putative ESE-1 binding site (EBS) was mapped within conserved region 2. Site-directed mutagenesis of the putative EBS abrogated the repressive effect of ESE-1 on NANOG promoter activity. ESE-1 directly interacted with the putative EBS to regulate transcriptional activity of NANOG. Furthermore, NANOG-induced proliferation and colony formation of NCCIT cells were inhibited by ESE-1 overexpression and stimulated by ESE-1 shRNA-mediated knockdown. Altogether, our results suggest that ESE-1 exerts an anti-proliferative effect on NCCIT cells by acting as a novel transcriptional repressor of NANOG.

1,25-dihydroxyvitamin D3 signaling-induced decreases in IRX4 inhibits NANOG-mediated cancer stem-like properties and gefitinib resistance in NSCLC cells

Recent studies have demonstrated that acquisition of cancer stem-like properties plays an essential role in promoting epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) resistance in non-small cell lung cancer (NSCLC); however, how to regulate cancer stem-like properties and EGFR-TKI resistance is largely unclear. In this study, we discovered that increased iroquois-class homeodomain protein 4 (IRX4) was related to gefitinib resistance in NSCLC cells. Knockdown of IRX4 inhibited cell proliferation, sphere formation, and the expression of CD133, ALDH1A1, NANOG, Sox2 and Notch1, and the transcriptional activity of NANOG promoter. IRX4 overexpression increased the protein level of NANOG and CD133 in PC-9 cells. Combination of knocking-down IRX4 with gefitinib increased cell apoptosis and decreased cell viability and the expression of p-EGFR and NANOG in PC-9/GR cells. IRX4 knockdown in a PC-9/GR xenograft tumor model inhibited tumor progression and the expression of NANOG and CD133 more effectively than single treatment alone. Knockdown of NANOG inhibited the expression of CD133 and restored gefitinib cytotoxicity, and NANOG overexpression-induced cancer stem-like properties and gefitinib resistance could be obviously reversed by knocking-down IRX4. Further, we found that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) reduced obviously the expression of IRX4 and NANOG by inhibiting the activation of TGF-β1/Smad3 signaling pathway; moreover, combination of 1,25(OH)2D3 and gefitinib decreased cell viability and proliferation or tumor progression and the expression of IRX4 and NANOG compared with single treatment alone both in PC-9/GR cells and in a PC-9/GR xenograft tumor model. These results reveal that inhibition of IRX4-mediated cancer stem-like properties by regulating 1,25(OH)2D3 signaling may increase gefitinib cytotoxicity. Combination therapy of gefitinib and 1,25(OH)2D3 by targeting IRX4 and NANOG, could provide a promising strategy to improve gefitinib cytotoxicity.

TRRAP stimulates the tumorigenic potential of ovarian cancer stem cells

Ovarian cancer is the most fatal gynecological malignancy in women and identification of new therapeutic targets is essential for the continued development of therapy for ovarian cancer. TRRAP (transformation/transcription domain-associated protein) is an adaptor protein and a component of histone acetyltransferase complex. The present study was undertaken to investigate the roles played by TRRAP in the proliferation and tumorigenicity of ovarian cancer stem cells. TRRAP expression was found to be up-regulated in the sphere cultures of A2780 ovarian cancer cells. Knockdown of TRRAP significantly decreased cell proliferation and the number of A2780 spheroids. In addition, TRRAP knockdown induced cell cycle arrest and increased apoptotic percentages of A2780 sphere cells. Notably, the mRNA levels of stemness-associated markers, that is, OCT4, SOX2, and NANOG, were suppressed in TRRAP-silenced A2780 sphere cells. In addition, TRRAP overexpression increased the mRNA level of NANOG and the transcriptional activity of NANOG promoter in these cells. Furthermore, TRRAP knockdown significantly reduced tumor growth in a murine xenograft transplantation model. Taken together, the findings of the present study suggest that TRRAP plays an important role in the regulation of the proliferation and stemness of ovarian cancer stem cells.

NANOG gene expression is regulated by the ETS transcription factor ETV4 in human embryonic carcinoma NCCIT cells

We demonstrated that ETV4 is a transcriptional activator of the NANOG gene in human embryonic carcinoma NCCIT cells. The endogenous expression of NANOG and ETV4 in naïve cells was significantly down-regulated upon differentiation and by shRNA-mediated knockdown of ETV4. NANOG transcription was significantly upregulated by ETV4 overexpression. A putative ETS binding site (EBS) is present in the region (−285 to −138) of the proximal promoter. Site-directed mutagenesis of the putative EBS (−196AGGATT−191) abolished NANOG promoter activity and ETV4 interacted with this putative EBS both in vivo and in vitro. Our data provide the molecular details of ETV4-mediated NANOG gene expression.

Characterization of the Nanog 5'-flanking Region in Bovine.

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5′-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5′-flanking sequences revealed that the −134 to −19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

The synergistic effect of 5Azadc and TSA on maintenance of pluripotency of chicken ESCs by overexpression of NANOG gene.

NANOG is a transcription factor that functions in embryonic stem cells (ESCs) and a key factor in maintaining pluripotency. Here, we cloned the NANOG gene promoter from the Rugao yellow chicken and constructed a dual luciferase reporter vector to detect its transcriptional activity and analyze the effects of 5-aza-2'-deoxycytidine (5-Azadc) and trichostatin A (TSA) on NANOG promoter activity and ESC pluripotency maintenance in vitro. NANOG transcriptional activity was enhanced when 5-Azadc and TSA were used alone or together, suggesting the possibility of elevated methylation of the CpG island in the NANOG regulatory region. When ESCs were cultured in basic medium with 5-Azadc and TSA in vitro, significantly more cell colonies were maintained in the 5-Azadc + TSA group than in the control group, which had many differentiated cells and few cell colonies after 6d of induction. On the tenth day of induction, the cells in the control group fully differentiated and no cell colonies remained, but many cell colonies were present in the 5-Azadc + TSA group. The expression of NANOG in the cell colonies was confirmed by indirect immunofluorescence. Furthermore, ESCs could be passaged to the 12th generation under 5-Azadc and TSA treatment and maintained their pluripotency. Thus, we showed that 5-Azadc and TSA can effectively maintain chicken ESC pluripotency in vitro by increasing NANOG gene expression.

Characterization of Bovine <i>NANOG</i>5’-flanking Region during Differentiation of Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (−420/+181) bovine NANOG 5′-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (−420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

GATA-1 directly regulates Nanog in mouse embryonic stem cells

Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression.

Bmi1 regulates self-renewal and epithelial to mesenchymal transition in breast cancer cells through Nanog.

BackgroundThe Bmi1 polycomb ring finger oncogene, a transcriptional repressor belonging to the Polycomb group of proteins plays an important role in the regulation of stem cell self-renewal and is elevated in several cancers. In the current study, we have explored the role of Bmi1 in regulating the stemness and drug resistance of breast cancer cells.MethodsUsing real time PCR and immunohistochemistry primary breast tissues were analyzed. Retro- and lentiviruses were utilized to overexpress and knockdown Bmi1, RT-PCR and Western blot was performed to evaluate mRNA and protein expression. Stemness properties were analyzed by flow cytometry and sphere-formation and tumor formation was determined by mouse xenograft experiments. Dual luciferase assay was employed to assess promoter activity and MTT assay was used to analyze drug response.ResultsWe found Bmi1 overexpression in 64% of grade III invasive ductal breast adenocarcinomas compared to normal breast tissues. Bmi1 overexpression in immortalized and transformed breast epithelial cells increased their sphere-forming efficiency, induced epithelial to mesenchymal transition (EMT) with an increase in the expression of stemness-related genes. Knockdown of Bmi1 in tumorigenic breast cells induced epithelial morphology, reduced expression of stemness-related genes, decreased the IC50 values of doxorubicin and abrogated tumor-formation. Bmi1-high tumors showed elevated Nanog expression whereas the tumors with lower Bmi1 showed reduced Nanog levels. Overexpression of Bmi1 increased Nanog levels whereas knockdown of Bmi1 reduced its expression. Dual luciferase promoter-reporter assay revealed Bmi1 positively regulated the Nanog and NFκB promoter activity. RT-PCR analysis showed that Bmi1 overexpression activated the NFκB pathway whereas Bmi1 knockdown reduced the expression of NFκB target genes, suggesting that Bmi1 might regulate Nanog expression through the NFκB pathway.ConclusionsOur study showed that Bmi1 is overexpressed in several high-grade, invasive ductal breast adenocarcinomas, thus supporting its role as a prognostic marker. While Bmi1 overexpression increased self-renewal and promoted EMT, its knockdown reversed EMT, reduced stemness, and rendered cells drug sensitive, thus highlighting a crucial role for Bmi1 in regulating the stemness and drug response of breast cancer cells. Bmi1 may control self-renewal through the regulation of Nanog expression via the NFκB pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-785) contains supplementary material, which is available to authorized users.

A Genome-wide RNAi Screen Identifies Opposing Functions of Snai1 and Snai2 on the Nanog Dependency in Reprogramming

Nanog facilitates embryonic stem cell self-renewal and induced pluripotent stem cell generation during the final stage of reprogramming. From a genome-wide small interfering RNA screen using a Nanog-GFP reporter line, we discovered opposing effects of Snai1 and Snai2 depletion on Nanog promoter activity. We further discovered mutually repressive expression profiles and opposing functions of Snai1 and Snai2 during Nanog-driven reprogramming. We found that Snai1, but not Snai2, is both a transcriptional target and protein partner of Nanog in reprogramming. Ectopic expression of Snai1 or depletion of Snai2 greatly facilitates Nanog-driven reprogramming. Snai1 (but not Snai2) and Nanog cobind to and transcriptionally activate pluripotency-associated genes including Lin28 and miR-290-295. Ectopic expression of miR-290-295 cluster genes partially rescues reprogramming inefficiency caused by Snai1 depletion. Our study thus uncovers the interplay between Nanog and mesenchymal factors Snai1 and Snai2 in the transcriptional regulation of pluripotency-associated genes and miRNAs during the Nanog-driven reprogramming process.

Nanog expression is negatively regulated by protein kinase C activities in human cancer cell lines

Nanog is a transcription factor that is essential for the maintenance of pluripotency of the embryonic stem cells. Nanog has been shown to be expressed in various kinds of human tumors, suggesting a role in tumorigenesis. In this study, we found that Nanog expression was upregulated by inhibition of protein kinase C (PKC) activity in six human cancer cell lines examined. In a Nanog non-expressing human nasopharyngeal carcinoma cell line, NPC-076, Nanog mRNA level and protein level were both induced and dose-dependently promoted by exposure to PKC inhibitors. Knockdown experiments showed that PKCα and PKCδ were two subtypes exerted most of the effect. The reporter assay showed that Nanog promoter activity was promoted by exposure of the cells to PKC inhibitors and the effect was dependent on the presence of the Octamer-Sox composite element. The involvement of Octamer-Sox composite element was further supported by the observation that silencing of Oct4 and Sox2 in NPC-076 cells attenuated the effects of PKC inhibitors. In Nanog-expressing human embryonal carcinoma cell lines, NT2/D1 and NCCIT, Nanog expression was suppressed by exposure to PKC activator Phorbol-12-myristate-13-acetate (PMA). Further study showed that overexpression of PKCα elicited a repressive effect on Nanog expression in NT2/D1 cells. Consistently, mutation of the Octamer-Sox composite element abolished the suppressive effect by PKC activator. Nanog expression was of cellular significance in that ectopic expression in NPC-076 stimulated cell proliferation and knockdown of the endogenous Nanog expression in NT2/D1-suppressed cell proliferation.

Abstract 2656: Using Nanog expression to identify breast cancer stem cell populations.

Abstract Many tumors have been demonstrated to contain a subpopulation of cancer stem cells (CSC) whose epigenetic modifications convey resistance to conventional therapies. Furthermore, tumor expression of genes associated with pluripotent embryonic cells, such as Oct-4, Sox-2 and Nanog, often correlates with poor prognosis. We hypothesized that expression of transcription factors that are required for induction and maintenance of pluripotency (Oct-4, Sox-2 and Nanog) would be a possible alternative to cell surface phenotype for identification of CSC populations. Therefore we evaluated Nanog expression in 4T1.2 murine breast carcinoma cells, as a biomarker for CSC sub-populations. Introduction of a Nanog promoter reporter that drives destabilized GFP into 4T1.2 cells resulted in a stable expression of GFP in about 1% of the cells. These GFP+ cell could be easily isolated by FACS cell sorting. On subsequent in vitro cell culture of GFP+ populations, up to 80% of the cells became GFP- over a period of 4 weeks. This would be consistent with a subsequent differentiation of these cells. By contrast GFP- populations remained 100% GFP-. In vitro CSC surrogate assays show that Nanog-GFP+ cells produced more spheroids in soft agar and under non-adherent growth conditions. More importantly, in vivo cell transfer studies demonstrated that Nanog-GFP+ cells were more efficient at generating experimental lung metastases when compared to Nanog-GFP-cells. Interestingly Nanog-GFP+ cells were more resistant to the chemotherapeutic drugs paclitaxel and bortezomib. However targeting the extrinsic apoptosis pathway with a combination of bortezomib and agonist antibodies to the TRAIL death receptor DR5 was equally effective against both Nanog-GFP+ and Nanog-GFP- cells. These findings suggest that Nanog promoter activity is a robust marker of highly metastatic 4T1.2 CSC subpopulations, and suggests that eradication of such cells may be required for improved anti-cancer therapies. Funded by NCI Contract HHSN261200800001E Citation Format: Alan D. Brooks, Rachel L. de Kluyver, Jimmy K. Stauffer, Marcella Kaddoura, Thomas J. Sayers. Using Nanog expression to identify breast cancer stem cell populations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2656. doi:10.1158/1538-7445.AM2013-2656

Optimal Ratio of Transcription Factors for Somatic Cell Reprogramming

Somatic cell reprogramming is achieved by four reprogramming transcription factors (RTFs), Oct3/4, Sox2, Klf4, and c-Myc. However, in addition to the induction of pluripotent cells, these RTFs also generate pseudo-pluripotent cells, which do not show Nanog promoter activity. Therefore, it should be possible to fine-tune the RTFs to produce only fully pluripotent cells. For this study, a tagging system was developed to sort induced pluripotent stem (iPS) cells according to the expression levels of each of the four RTFs. Using this system, the most effective ratio (Oct3/4-high, Sox2-low, Klf4-high, c-Myc-high) of the RTFs was 88 times more efficient at producing iPS cells than the worst effective ratio (Oct3/4-low, Sox2-high, Klf4-low, c-Myc-low). Among the various RTF combinations, Oct3/4-high and Sox2-low produced the most efficient results. To investigate the molecular basis, microarray analysis was performed on iPS cells generated under high (Oct3/4-high and Sox2-low) and low (Oct3/4-low and Sox2-high) efficiency reprogramming conditions. Pathway analysis revealed that the G protein-coupled receptor (GPCR) pathway was up-regulated significantly under the high efficiency condition and treatment with the chemokine, C-C motif ligand 2, a member of the GPCR family, enhanced somatic cell reprogramming 12.3 times. Furthermore, data from the analysis of the signature gene expression profiles of mouse embryonic fibroblasts at 2 days after RTF infection revealed that the genetic modifier, Whsc1l1 (variant 1), also improved the efficiency of somatic cell reprogramming. Finally, comparison of the overall gene expression profiles between the high and low efficiency conditions will provide novel insights into mechanisms underlying somatic cell reprogramming.

Bone morphogenetic protein-7 regulates Snail signaling in carbon tetrachloride-induced fibrosis in the rat liver

The aim of this study was to explore the molecular mechanism of the bone morphogenetic protein-7 (BMP-7) downregulation of Snail-mediated E-cadherin repression and mesenchymal-epithelial transition (MET) induction, since little is presently known about this issue. In this study, our aim was to elucidate the underlying mechanism by which cells acquire liver fibrosis characteristics after epithelial-mesenchymal transition (EMT). Cell cultures were exposed to Snail alone or in the presence of BMP-7; control cultures were exposed to medium only. The expression of the mRNA encoding α-smooth muscle actin (α-SMA), Snail and E-cadherin in rat liver epithelial cells was determined by real-time quantitative PCR (RT-PCR) and the main results were confirmed by ELISA. Cell differentiation was determined by analysis of the expression of α-SMA, Snail and E-cadherin by western blotting and co-immunoprecipitation. We demonstrated Snail-induced upregulation of mRNAs encoding α-SMA and downregulation of mRNAs encoding E-cadherin in rat liver epithelial cells when compared with unstimulated cells, and confirmed these results at the protein level. BMP-7 downregulated Snail-induced α-SMA and upregulated E-cadherin release compared with untreated and Snail-treated cells. In summary, we demonstrated that BMP-7 induces MET through decreased downregulation of Snail. In addition, Snail1 directly regulates Nanog promoter activity. Notch signaling is also involved in this process.

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.

A Tcf/Lef element within the enhancer region of the human NANOG gene plays a role in promoter activation

NANOG is a homeodomain-containing transcription factor that is essential for the maintenance of pluripotency and self-renewal in embryonic stem cells. However, the molecular mechanisms underlying the regulation of NANOG expression in human cells remain largely unknown. Here, we investigated the role of Tcf/Lef response elements located in the enhancer of the human NANOG gene. We found that forced expression of Lef1 or β-catenin stimulated human NANOG promoter activity, while shRNA-mediated knockdown of β-catenin reduced Lef1-induced NANOG promoter activation. Deletion or mutation of the Tcf/Lef element within the enhancer region of the human NANOG gene completely abrogated Lef1-induced NANOG promoter activity. The results of a chromatin immunoprecipitation assay demonstrated that Lef1 and β-catenin bind to the Tcf/Lef element in the enhancer region of the NANOG gene. Forced expression of GSK-3β inhibited basal, Lef1-induced, and β-catenin-induced NANOG promoter activity, while treatment with the GSK-3β inhibitor SB216763 resulted in the accumulation of β-catenin and NANOG protein. Furthermore, Dvl-1-induced NANOG promoter activity was abrogated by the expression of β-catenin shRNA. Stable overexpression of Dvl-1 caused β-catenin and NANOG to accumulate. These results indicate that the Tcf/Lef response element in the enhancer region of the human NANOG gene is able to stimulate NANOG gene transcription.

Abstract 3349: Nanog-expressing breast cancer tumor initiating cells (TICs) are highly metastatic and are resistant to the effects of proteasome inhibition

Abstract Functional heterogeneity of transformed cells exists within a single tumor mass and between distinct tumor nodules. Many tumors have also been demonstrated to contain a subpopulation of tumor initiating cells (TICs) whose epigenetic modifications convey resistance to conventional therapies. It has been hypothesized that TICs represent the terminal product of epithelial-mesenchymal transition which is linked to metastatic potential. Therefore we examined the metastatic potential of TICs in the 4T1 murine breast carcinoma model, and evaluated Nanog expression as a biomarker for highly metastatic 4T1 sub-populations. Traditionally TICs have been identified and isolated through expression of selected cell surface markers and functional assays, however cell surface phenotype alone fails to identify CSC in all tumors from the same organ or in all patients. We hypothesized that expression of transcription factors that are required for induction and maintenance of pluripotency (Oct-4, Sox-2 and Nanog) could be superior to cell surface phenotype for identification of TICs. Thus we adapted a commercial promoter reporter system where the Nanog promoter drives copepod GFP which has been destabilized to better reflect rapid changes in transcriptional activity. I ntroduction of a Nanog promoter reporter construct into 4T1.2 cells resulted in stable expression of GFP in about 0.5% of the cells. In vivo tumor transfer studies confirmed that Nanog promoter activity (4T1.2-GFPpos cells) was associated with a significantly enhanced ability to form experimental lung metastases. In addition, in vitro TIC surrogate assays showed that 4T1.2 GFPpos cells produced increased numbers of spheroids in both soft agar and under non-adherent growth conditions. Finally Nanog-GFPpos cells were more resistant to killing by Paclitaxel than Nanog-GFPneg cells, but were more sensitive to Salinomycin. Together these findings suggest that Nanog promoter activity is a robust marker of metastatic 4T1.2 TICs. We have previously observed that administration of the proteasome inhibitor Bortezomib (Bzb) has moderate anti-4T1.2 tumor activity in vivo therefore we examined the sensitivity of the various 4T1.2 populations to Bzb treatment in vitro. 4T1.2 Nanog-GFPpos cells were resistant to the cytostatic effects of Bzb while 4T1.2 Nanog-GFPneg cells were sensitive. The molecular basis of this difference is under investigation, but is not due to differences in accumulation of ubiquitinated proteins. Taken together, these findings show that Nanog promoter activity is a marker of highly metastatic TICs, and suggest that eradication of TICs may be required for curative anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3349. doi:10.1158/1538-7445.AM2011-3349

The Pluripotency Regulator Zic3 Is a Direct Activator of the Nanog Promoter in ESCs

The transcription factor Zic3 is required for maintenance of ESC pluripotency. By genome-wide chromatin immunoprecipitation (ChIP-chip) in ESCs, we have identified 379 direct Zic3 targets, many of which are functionally associated with pluripotency, cell cycle, proliferation, oncogenesis, and early embryogenesis. Through a computational analysis of Zic3 target sequences, we have identified a novel Zic3 consensus binding motif (5'-CC(C)/(T) GCTGGG-3'). ChIP results and in vitro DNA binding assays revealed that Zic3 binds with high affinity and specificity on the Nanog promoter. Here, we demonstrate that Zic3 functions as a transcriptional activator of the Nanog promoter in three ways: (a) Nanog transcript levels are sustained with Zic3 overexpression in differentiating ESCs, (b) Zic3 depletion in ESCs downregulates Nanog promoter activity, and (c) Zic3 overexpression leads to increased Nanog promoter activity. Furthermore, the activity of a mutant Nanog promoter with ablated Oct4/Sox2 binding is rescued by Zic3 overexpression to nearly wild-type levels. This indicates that Nanog is a positive transcriptional target of Zic3 in a mechanism that is independent of Oct4/Sox2 binding. Hence, we demonstrate an important pathway for regulation of Nanog expression in pluripotent ESCs through direct activation by Zic3.

Abstract 4249: Shape change induces nitric oxide and stemness in colorectal cancer cells

Abstract Shape changes in cancer cells profoundly alter the expression of genes that affect malignant properties. We postulated that shape change may increase expression of NANOG, an embryonic stem cell transcription factor expressed in human colorectal carcinoma (CRC) that regulates pluripotency and proliferation. Since shape change also increases intracellular nitric oxide (iNO) expression, we further postulated that iNO modulates NANOG expression. Clone A and CX-1 human CRC lines were cultured in serum-containing medium in monolayer cultures or in spheroids in serum free medium in low attachment plates. NANOG promoter activity was assessed in stable transfectants expressing promoter-driven GFP. iNO concentrations were determined by fluorescence cytometry with DAF-FM diacetate while Nanog gene and protein expression was analyzed by qRT-PCR and densitometry of western blots, respectively. Spherogenicity was measured by the percentage of spheroids formed from single cells in serum-free medium. Monolayer cultures with flat, spreading cells had low NANOG gene and protein expression, promoter activity and iNO whereas spheroids displayed high NANOG gene expression, promoter activity and iNO. 7 day monolayer cultures initiated with 104 cells/ml (Sparse) grew horizontally compared to cultures initiated with 106 cells/ml (Dense) that grew vertically and formed colonies. Sparse cultures had low NANOG gene expression, low NANOG promoter activity and low iNO. Dense cultures had high NANOG gene expression, NANOG promoter activity and iNO. Since NO stimulates cGMP production, the effects of the NO synthase inhibitor L-NMMA, NOC (a NO donor), 8-Br-cGMP (cGMP agonist) and ODQ (cGMP inhibitor) were tested on CRC monolayers. Physiologic concentrations of NOC increased NANOG and Oct4 gene and protein expression 3-fold in highly metastatic CX-1 cells but not in weakly metastatic Clone A cells. NOC also increased CD44 in CX-1 but not Clone A. cGMP may regulate NANOG expression since 8-Br-cGMP increased NANOG and Oct4 protein expression up to 3-fold in CX-1 but not in Clone A and without affecting CD44 expression. ODQ inhibited NANOG expression. L-NMMA stimulated both NANOG protein expression and the spherogenicity of CX-1 but not Clone A. NOC and 8-Br-cGMP increased inducible NO synthase (NOS2) but not NOS3 gene expression 3-fold in both spheroids and monolayers while ODQ decreased NOS2 levels. Assays are in progress to test the effects of NOC and the cGMP modulators on spherogenicity. Shape change, iNO and cGMP enhance NANOG, NOS2 and CD44 expression to alter CRC cancer stem cell function. cGMP may be an important regulator of stemness genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4249.

Abstract 4248: NANOG depletion decreases cell proliferation rate in colorectal carcinoma cells and tumor growth in nude mice

Abstract Nanog is a critical transcription factor involved in maintaining embryonic stem cells in a pluripotent, dedifferentiated state and expression of NANOG or its nearly identical retrogene NANOGP8 is increased in human gastric, hepatocellular, and colon cancer. Our postulate is that NANOG/NANOGP8 is critical for the functions of putative CSC in colorectal carcinoma (CRC): self-renewal, pluripotency, spherogenicity, tumorigenicity and resistance to chemotherapy. Five human CRC lines are being used in this study (HCC 2998, KM-12c, Clone A, MIP-101, CX-1). CX-1 cells are more aggressive than the other CRC because they form more spheroids from single cells and are highly metastatic after intrasplenic compared to the other CRC that are tumorigenic but weakly metastatic. qRT-PCR analysis of spheroids of CX-1 and Clone A cultured in suspension in serum free medium indicated that NANOG transcript levels were increased by 43- and 12-fold, respectively, relative to monolayer cultures while the relative increase in transcript levels of several cancer stem cell markers such as EPCAM, ALDH1A1, ALDH3A1, CD44, CD133, and CD166 was less than 5-fold except for POU5F1 and SOX2 in Clone A, which were 5- and10-fold respectively. NANOG promoter activity was low in monolayer culture in CRC stable transfectants with a GFP NANOG promoter, but consistently increased as CRC detached and formed spheroids in serum free media. Stable transfection with lentiviral NANOG shRNA decreased NANOG transcript levels and spherogenicity of Clone A and CX1 cells compared to vector controls in a single cell spherogenicity assay in serum-free medium. NANOG downregulation also induced a S phase cell cycle arrest with inhibition of ATM and WEE1 expression with associated reduction in phosphorylation of other key cell-cycle checkpoint proteins. Finally, marked tumor growth retardation was observed in NOD/SCID mice injected subcutaneously with NANOG shRNA transfected cells compared to parental or vector controls. NANOG shRNA transfected cells were also more resistant to treatment with Topotecan, a camptothecin. In summary, our data suggest that expression of NANOG/NANOGP8 occurs in human CRC and may be critical for such functions of putative CSC as spherogenicity, cell proliferation, tumor formation and resistance to chemotherapy Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4248.