Author contribution: C.H.W.: collection and assemble the data, data analysis and interpretation, manuscript writing, and final approval of the manuscript; N.M., L.F.L., and S.Y.C.: manuscript writing and final approval of the manuscript; Y.T.L.: collection and assemble the data, data analysis and interpretation, and final approval of the manuscript; C.C.W.: collection and assemble the data and final approval of the manuscript; M.H.: provision study material and final approval of the manuscript; C.C.Y.: collection and assemble the data and data analysis and interpretation; J.L.: data analysis and interpretation, manuscript writing, and final approval of the manuscript. C.-H.W., N.M., and Y.-T. Lin contributed equally to this article.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS August 16, 2012.
In contrast to the somatic cells, embryonic stem cells (ESCs) are characterized by its immortalization ability, pluripotency, and oncogenicity. Revealing the underlying mechanism of ESC characteristics is important for the application of ESCs in clinical medicine. We performed systematic functional screen in mouse ESCs with 4,801 shRNAs that target 929 kinases and phosphatases. One hundred and thirty-two candidate genes that regulate both ESC expansion and stem cell marker expression were identified. Twenty-seven out of the 132 genes were regarded as most important since knockdown of each gene induces morphological changes from undifferentiated to differentiated state. Among the 27 genes, we chose nonmetastatic cell 6 (Nme6, also named as Nm23-H6) and nonmetastatic cell 7 (Nme7, also designated as Nm23-H7) to study first. Nme6 and Nme7 both belong to the members of nucleoside diphosphate kinase family. We demonstrate that Nme6 and Nme7 are important for the regulation of Oct4, Nanog, Klf4, c-Myc, telomerase, Dnmt3B, Sox2, and ERas expression. Either knockdown of Nme6 or Nme7 reduces the formation of embryoid body (EB) and teratoma. The overexpression of either Nme6 or Nme7 can rescue the stem cell marker expression and the EB formation in the absence of leukemia inhibiting factor. This implies the importance of Nme6 and Nme7 in ESC renewal. This finding not only pinpoints Nme6 or Nme7 can regulate several critical regulators in ESC renewal but also increases our understanding of the ESC renewal and oncogenesis. STEM Cells2012;30:2199–2211
Embryonic stem cells (ESCs) are derived from the inner cell mass of the early embryo and are characterized by the ability in immortalization, pluripotency, and teratoma formation. These characteristics distinguish ESCs from somatic cells and adult stem cells. Moreover, undifferentiated mouse ESCs can exit cell cycle and be induced into differentiated state by either removal of leukemia inhibiting factor (LIF) in the culture medium or suspension culture of ESCs to form embryoid body (EB). The EB expresses ectoderm, mesoderm, and endoderm markers. In vivo, subcutaneous injection of ESCs leads to teratoma formation that contains ectoderm, mesoderm, and endoderm. These results indicated the pluripotency and oncogenicity of ESCs. Fortunately, after differentiation, ESCs lose both oncogenicity and pluripotency. The progeny of ESCs can be used in regenerative medicine. Thus, the molecular mechanisms of ESC immortalization, pluripotency, differentiation, and teratoma formation become interesting issues to address.
LIF either acts in combination with bone morphogenesis protein or fetal bovine serum (FBS) to sustain mouse ESC growth [1, 2]. To maintain ESC renewal, LIF activates signal transducer and activator of transcription-3 (STAT3) through JAK kinase [3, 4]. Additionally, the inhibitors of glycogen synthase kinase-3 (GSK3)-beta and mitogen-activated protein kinase (MAPK) can maintain mouse ESC renewal in the absence of LIF . LIF, GSK3-beta, and MAPKs are representative membrane and cytoplasmic signal transducers that modulate ESC differentiation and self-renewal.
The core transcription factor regulatory circuit has been extensively studied in ESCs since it governs the stem cell fates. Several transcriptional regulators such as Oct4, Nanog, STAT3, Sox2, c-Myc, Esrrb, Klf4, Ronin, Tcl1, Tbx3, and Rest have been extensively investigated [6, 7]. Among them, transcriptional factors Oct4 and Nanog have been demonstrated to play important roles in ESC pluripotency, and inhibition of either Oct4 or Nanog expression forces ESCs differentiate into trophoblasts or extraembryonic endoderm [8–11]. Interestingly, several signals that are essential for ESC renewal have been shown to be important for oncogenesis, such as c-Myc is crucial for tumorigenesis, antiapoptosis, and stem cell renewal [12, 13]. ERas is both essential for the oncogenicity of ESCs and gastric cancer [14, 15]. Some transcriptional regulators play crucial roles in ESC characteristics. Either the combination of Oct4, Sox2, Myc, and Klf4, or Oct4, Nanog, Sox2, and Lin28 is sufficient to reprogram fibroblasts to induced pluripotent stem cells (iPSCs) [16–18]. iPSCs resembles ESCs in terms of immortalization, pluripotency, and oncogenicity. These studies provide information about how ESCs and iPSCs maintain pluripotency, oncogenesis, and self-renewal by regulating the transcriptional circuits and inhibiting the differentiation pathways with multiple transcriptional regulators.
The answer to how the ESC signals activate the core transcriptional factors to decide cell fate is still elusive. Kinases and phosphatases govern the process of cell proliferation, differentiation, apoptosis, and carcinogenesis, and are frequently served as the therapeutic targets for cancer therapy [19, 20]. They are the key upstream regulators of the transcriptional factors. For instance, the cytoplasmic kinase, phosphoinositide 3-kinase (PI3K), activates Nanog [21–23].
The activities of kinases and phosphatases were largely regulated by phosphorylation or conformational changes rather than the alteration at the transcriptional levels. As a result, it is not easy to efficiently pinpoint the crucial kinases/phosphatases responsible for stem cell functions from microarray or deep sequencing. Alternatively, systematic shRNA screening had been shown to be the approach to identify novel kinases/phosphatases that are crucial for stem cell pluripotency, renewal, and oncogenicity. Genome-wide RNAi screen in mouse ESC with pooled screening strategy has been performed using Oct4/telomerase as a reporter [24–26]. The screening based on other stem cell markers has not been described. To identify additional self-renewal modulators, we used the array-based screen with the alkaline phosphatase (ALP) assay, relative cell number, and morphological change as the reporter systems. Systematic kinase/phosphatase shRNA functional screen was performed by 4,081 shRNAs targeting 929 kinases and phosphatases to simultaneously identify multiple pivotal genes that regulate ESC proliferation and pluripotency.
In this shRNA functional screen, we identified that 27 genes induced morphological changes. Among the hits, Nme6 and Nme7 both belong to the members of nucleoside diphosphate kinase (NDPK) family. The Nme family is involved in various molecular processes and act differently across the cancer cell types studied . We further demonstrate that nonmetastatic cell 6 (Nme6) and nonmetastatic cell 7 (Nme7) are important for ESC renewal, pluripotency, and oncogenesis.
MATERIALS AND METHODS
All the reagents for cell culture and quantitative real-time polymerase chain reaction (QRT-PCR), otherwise unless specified, were purchased from Invitrogen (Grand Island, NY, USA, http://www.invitrogen.com/). All the chemicals, otherwise unless specified, were obtained from Sigma (St. Louis, MO, USA, http://www.sigmaaldrich.com/united-states.html/). All the procedures practiced with recombinant DNA follow the National Institutes of Health guidelines.
Mouse ESC lines D3 (ATCC, Manassas, VA, USA, http://www.atcc.org/) and EBRTch3 (RIKEN, Ibaraki, Japan, http://www.riken.jp/engn/) were maintained on inactivated STO cells (ATCC). The culture medium is Dulbecco's modified Eagle's medium (DMEM) supplied with 15% of FBS (Hyclone, Logan, UT, USA, http://www.thermofisher.com/global/en/home.asp/), 103 unit/ml LIF (Chemicon, Billerica, MA, USA, http://www.millipore.com/), 0.1 mM nonessential amino acid, 1 mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mM β-mercaptoethanol (Sigma). For feeder-free culture, cells were cultured with the same medium, but the FBS was replaced by knockout serum.
Array-Based High-Content Screening
All shRNA plasmids and viruses were purchased form National RNAi Core Facility (Taipei, Taiwan, http://rnai.genmed.sinica.edu.tw/index/). AlamarBlue (AB) and ALP assay were performed at day 5 after virus infection by a liquid handling system with multiplicity of infection = 1 (Freedom EVO, Tecan, Männedorf, Switzerland, http://www.tecan.com/). Ten microliters of AB (Biotium, Hayward, CA, USA, http:// www.biotium.com/) was added to each well and incubated with cells for 2 hours at 37°C. The absorbance at OD 570 nm and 600 nm was measured. Cells were fixed by methanol for 1 minute, after PBS washing, then 70 μl p-nitrophenyl phosphate liquid substrate was added to cells and incubated for 10 minutes at room temperature. The ALP activity was detected by the absorption at OD 405 nm wavelength.
EB was formed by hanging drop method. Cells were cultured in the feeder-free culture medium with/without LIF. Then cells were suspended in EB medium that contains DMEM supplied with 10% FBS and 1 mM L-glutamine. Cell concentration was adjusted to 200 cells per drop (20 μl). After 3 days, the size and number of EB in each plate were quantified.
ALP Activity Assay
Cells were fixed by methanol for 1 minute. For ALP staining, Alkaline Phosphatase Detection Kit (Millipore) was performed as manufacture's instruction.
Total RNA was isolated using TRIzol reagent. cDNA was generated by Superscript III with random primers. QRT-PCR reaction was performed using SyBr Green reaction mixture (Kapa Biosystems, Woburn, MA, USA, www.kapabiosystems.com) with ABI 7000 Prism Real-Time PCR system (Life Technologies Corporation, Carlsbad, CA, USA, http://www.appliedbiosystems.com/absite/us/en/home.html/). The primer sequences are listed at Supporting Information Table S1. All QRT-PCR reactions were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Plasmid Construction and Transfection
The cDNA of Nme6, Nme7v1, and Nme7v2 was amplified to form D3 cells and then constructed into pEF-DEST51 vector (Invitrogen). For transient expression in mouse ESCs, pNme6, pNme7v1, pNme7v2, or control pGFP was transfected by Lipofectamine 2000 (Invitrogen). After 5 hours, 1 μg/ml puromycin was added for selection. For the generation of ROSA26 knockin stable lines, the cDNAs of Nme6, Nme7v1, and control eGFP were reconstructed into exchange vector of ROSA-TET system (kindly provided by Dr. Shinji Masui, Tokyo, Japan) . The recombination reaction of the TET-regulatable unit into the ROSA locus was performed as previously described .
Cells were lysed with 1% NP40 lysis buffer (1% NP40, 50 mM Tris pH = 8, 150 mM NaCl, 2 mM EDTA, and 1 mM Na3VO4) with complete protease inhibitor (Roche, Upper Bavaria, Germany, http://www.roche.com/index.htm/). Western blot was performed with anti-actin (Sigma, A5441), anti-HA (Covance, MMS-101R, Princeton, NJ, USA, www.covance.com), anti-Oct4 (Santa Cruz, sc-5279, Santa Cruz, CA, USA, http://www.scbt.com/), anti-Nanog (Cell signaling, 3580, Danvers, MA, USA, http://www.cellsignal.com/), anti-c-Myc (Santa Cruz, sc-764), anti-ERas (Santa Cruz, sc-51072), anti-Sox2 (Santa Cruz, sc-20088), anti-HP1 (Santa Cruz, sc-28735), and anti-α-tubulin (Sigma, T5168) antibodies. The membranes were incubated with SuperSignal ELISA Femto Maximum Sensitivity Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA, http://www.thermofisher.com) and then imaged with LAS-4000 system (FujiFilm, Tokyo, Japan, http://www.fujifilm.com/). The contrast of the whole image was linearly adjusted by Fuji Film Multi Gauge, version 3.0 (FujiFilm).
Cells were fixed by 4% paraformaldehyde in phosphate buffer saline (PBS) for 20 minutes RT and treated with 0.1% Triton X-100 for 10 minutes. After PBS washing, cells were incubated with anti-Sox1 (Cell Signaling, 4194) or anti-FoxA2 antibody (Santa Cruz, sc-9187) (2% bovine serum albumin (BSA) in PBS) overnight at 4°C. Then cells were further incubated with Alexa Fluor 488 anti-rabbit IgG/anti-goat IgG (Invitrogen). The nuclear were stained with 4′,6-diamidino-2-phenylindole (1 μg/ml).
Extraction of Nuclear and Cytoplasmic Fraction
The extraction of cytoplasmic and nuclear fraction was performed by manufacturer's manual (Thermo Fisher Scientific, Waltham, MA, USA). In brief, cells pellets were washed with lysis buffer. The supernatants were collected as cytoplasmic fraction, whereas the nuclear-containing pellet was further treated with nuclear washing buffer. After centrifugation, the remaining pellet was mixed with nuclear storage buffer. Finally, the pellet was reacted with nuclei lysis reagent. Both nuclear and cytoplasmic fractions were assayed by Western blot analysis.
Luciferase Reporter Assay
Luciferase reporter plasmid driven by the Oct4 upstream promoter region is constructed by inserting the Oct4 upstream promoter region (−2,173 to −1) into the pGL3-basic (Promega) (kindly provided by Prof. Fuyuki Ishikawa at Kyoto University, Japan). Cells were cultured in 12-well plates and cotransfected with the corresponding luciferase reporter vector (500 ng per well) and pRL-TK (100 ng per well) in the presence of pEF-DEST51-Nme7 (500 ng per well) or shNme7 plasmids (500 ng per well) using Lipofectamine 2000. Cells were selected by either 3 μg/ml blasticidin (for overexpression of Nme7) or 1 μg/ml puromycin (knockdown of Nme7). All the cells were harvested 48 hours postselection and assay for luciferase activity using the Luciferase reporter assay system (Promega, Madison, WI, USA, http://www.promega.com/) following manufacturer's instructions. Samples were measured using a VICTOR3 luminometer (PerkinElmer Technologies, Waltham, MA, USA, http://www.perkinelmer.com/). The luciferase activities were then normalized to the measurements of renilla luciferase assay.
Identification of Pluripotency-Related Genes by Array-Based RNAi Screen in Mouse ESCs
To find the sensitive markers that can rapidly detect the changes of ESC pluripotency, several commonly used stem cell differentiation markers were monitored after the removal of LIF (Supporting Information Fig. S1A). At day 1, changes in cell morphology and colony size were observed. At day 2, the decrease of ALP activities was detected. At day 3, the decrease of nuclear size as well as downregulation of Oct4 was observed. At day 4, the rate of cell proliferation was decreased. Among them, ALP activity was chosen as the marker for the primary screening, due to its early response to differentiation (at day 2) and acceptable signal-to-noise ratio (Supporting Information Fig. S1B). It is of note that ALP also was the first marker presence during the reprogramming process of iPSCs. AB assay was used to measure the relative cell number (Supporting Information Fig. S1C). The ALP activities were normalized by AB activities. Unlike the previous screening system using Oct4 as the reporter, we have the advantage of discovering genes beyond Oct4 pathway.
To identify novel genes essential for ESC pluripotency, we used array-based RNAi screen to knock down specific genes. Totally, 4,801 shRNAs targeting 929 kinases or phosphatases were used for functional screen in the 96-well format (one shRNA per well). We identified 358 shRNAs targeting 132 genes resembled Oct4 shRNA in downregulating ALP/AB and AB activities (Fig. 1A). Candidate genes were defined as having Z-scores less than −1.5 (Fig. 1B and Supporting Information Table S2). To avoid the off-target effect, all selected genes were verified by at least two independent shRNAs. Two known pluripotency-related genes, PI3K and Oct4, were included in candidate gene list to demonstrate the efficacy of screen (Fig. 1B).
To select candidate genes that not only reduces stem cell marker but also induces ESC differentiation, we used morphological change as the second indicators for the screening (Fig. 1A). We classified the cell morphology of colonies into differentiated, partially differentiated, and undifferentiated types (Fig. 1C). The percentages of differentiated, partially differentiated, and undifferentiated colonies are shown in Figure 1C. Twenty-seven genes that were targeted by 59 shRNAs were selected based on their morphological changes after shRNA virus infection (Fig. 1A, 1C). Nme6 and Nme7 were further selected to pursue their roles in ESC renewal because they belong to the same family and showed prominent changes in our detection system.
Reduction of Either Nme6 or Nme7 Causes Mouse ESC Differentiation and the Downregulation of Pluripotency-Related Genes
In order to examine the roles of Nme6 or Nme7 in ESCs, shRNAs of Nme6 (shNme6-1 and shNme6-2) or shRNAs of Nme7 (shNme7-1 and shNme7-2) were, respectively, transfected into ESCs (Fig. 2A, 2B). shLuc and shOct4 were the negative and positive control for the morphological changes. The cells transfected with shNme6s or shNme7s became flat and their cell colonies were more spread out (Fig. 2B). In addition to the morphological change, the ESC colony numbers of both Nme6 and Nme7 shRNA-transfected cells were reduced as opposed to shLuc. The result suggested that the ability of cell expansion is also hampered in Nme6 or Nme7 knockdown ESCs (Fig. 2B). However, the knockdown of Nme6 or Nme7 did not affect the survival of mouse embryonic fibroblast (Supporting Information Fig. S2).
Since the inhibition of either Nme6 or Nme7 induces cell differentiation, we next investigated whether each of them modulates the key transcriptional regulators. The mRNA expression levels of 16 pluripotency and renewal genes were quantified by QRT-PCR (Fig. 2C, Supporting Information Fig. S3). We examined the expression of Oct4, Nanog, Sox2, Klf4, c-Myc, Dnmt1, Dnmt3a, Dnmt3B, telomerase (Tert), Rest, Lin28, Ronin, Tcl1, p53, ERas, and Tbx3. Interestingly, six stem cell key regulators, Oct4, Klf4, ERas, c-Myc, Tert, and Dnmt3B were reduced in both Nme6- and Nme7-knockdown ESCs (Fig. 2C). In contrast, Tcl1 may be regulated only by Nme6 but not by Nme7 (Supporting Information Fig. S3). This suggests that Nme6 and Nme7 may have different functions. The results of Western blot analysis confirm the changes of Oct4, c-Myc, and ERas in Nme6 or Nme7 knockdown transfectants (Fig. 2D). Interestingly, Nanog/Sox2 protein was downregulated in shNme6 or shNme7 transfectants, but the RNA level is unaffected (Fig. 2D, Supporting Information Fig. S3). We hypothesize that Nme6 and Nme7 can regulate Nanog/Sox2 at the post-transcriptional level. We observed that Nme6 and Nme7 are downregulated upon ESC differentiation into EB (Fig. 2E).
Overexpression of Either Nme6 or Nme7 Restores the Phenotypic Change Mediated by shNme6 or shNme7
To verify that the above results are from the loss of either Nme6 or Nme7 genes, but not from the off-target effect of shRNAs, Nme6 and Nme7 genes were overexpressed to rescue the expression. Nme7 has two splicing variants, named Nme7v1 (42.5 kDa) and Nme7v2 (31 kDa). The Nme7v1 has two NDPK domains, while Nme7v2 has only one NDPK domain. The overexpression of Nme6 or Nme7 could be detected by QRT-PCR. (Fig. 3A). Furthermore, by the cotransfection of Nme6, Nme7v1, or Nme7v2 expression plasmid, the expression of Nme6 and Nme7 can be successfully restored in shNme6- or shNme7-transfected ESCs (Fig. 3A). The ectopic expression of Nme6, Nme7v1, or Nme7v2 increased the AB activity in the shLuc, shNme6, or shNme7 transfectants, respectively (Fig. 3B). Interestingly, Nme6, Nme7v1, or Nme7v2 expression plasmid increased the ALP/AB activity up to threefold. Furthermore, these plasmids can fully rescue the expression of the stem cell marker ALP which were downregulated by either shNme6 or shNme7 (Fig. 3C, 3D). The result demonstrates that the overexpression of either Nme6 or Nme7 rescues the effects of shNme6 or shNme7.
Overexpression of Either Nme6 or Nme7 Rescues the Expression of Pluripotency-Related Genes in the Absence of LIF
LIF is one of the key cytokines to support ESC growth in the absence of feeder layer . To examine whether Nme6 and Nme7 can induce the pluripotency gene expression in the absence of LIF, in another ESC line (E14tg2), either Nme6 or Nme7 was knocked into ROSA26 locus with the ROSA-TET system. ROSA-TET system carries a tetracycline-inducible transgene at the Gt (ROSA) 26asSor (ROSA26) locus. The gene-specific integration at the ROSA locus is done via homologous recombination (Supporting Information Fig. S4A, S4B), thereby avoiding the random insertion mutagenesis. The knockin of the Nme6 or Nme7 gene in the ROSA26 locus was confirmed by PCR (Supporting Information Fig. S4C). The removal of tetracycline induced Nme6 or Nme7 expression (Fig. 4A, 4C). In the absence of LIF, the transcripts of Nanog, Oct4, Klf4, c-Myc, Tert, Dnmt3B, and ERas were all downregulated (Fig. 4B, 4D). However, the expression of these transcripts could be rescued by the overexpression of Nme6 or Nme7 in a dose-dependent manner (Fig. 4B, 4D). This result suggests Nme6 or Nme7 is sufficient to activate the expression of these pluripotency-related genes.
The Oct4, Klf4, and C-Myc Are the Early Responsive Genes of Nme6 or Nme7
Nme6 or Nme7 can regulate expression of Oct4, Klf4, c-Myc, ERas, telomerase, and Dnmt3B. To figure out Nme6 or Nme7 early responsive genes, we analyzed their mRNA levels at different days after ESC transfected with the shRNAs or cDNAs of Nme6 or Nme7. After shRNAs/cDNAs transfection, the mRNA levels of Oct4, Klf4, and c-Myc were altered at day 2 and were further reduced/enhanced at day 3. The regulation of Tert by shRNA of Nme6 or Nme7 occurred only after day 3 (Supporting Information Fig. S5A). Response of Dnmt3B was observed at day 2 and day 3 after the transfection of Nme6 or Nme7 shRNAs. However, it is not until day 3 that overexpressed Nme6 or Nme7 upregulated Dnmt3B (Supporting Information Fig. S5B). ERas transcripts were upregulated at day 2 after shRNA transfection, then significantly downregulated at day 3 (Supporting Information Fig. S5A), indicating that other factors might be involved in the control of ERas expression. However, when we overexpressed either Nme6 or Nme7, ERas transcripts increased significantly after day 2 and day 3 of transfection.
Reduction of Either Nme6 or Nme7 Expression Induces ESC Differentiation
To examine if either Nme6 or Nme7 blocks ESC differentiation, the expression levels of ectoderm, endoderm, and mesoderm markers were examined in shNme6- or shNme7-transfected cells. The knockdown efficiencies of shNme6-1, shNme6-2, shNme7-1, and shNme7-2 are 93%, 95%, 78%, and 52%, respectively (Fig. 5A, 5E). Ectoderm and endoderm markers such as Sox1, FoxA2, and GATA4 were upregulated in Nme6 or Nme7 knockdown cells (Fig. 5B, 5C, 5F, 5G). The knockdown of Nme7 but not Nme6 significantly increases the expression of the key mesoderm marker Brachyury (Fig. 5D, 5H). Additionally, the knockdown of either Nme6 or Nme7 increases the protein expression of Sox1 and FoxA2 demonstrated by immunofluorescence assay (Fig. 5I, 5J). The results suggest that Nme6 and Nme7 both maintain ESC pluripotency and block differentiation.
Overexpression of Either Nme6 or Nme7 Rescues EB Formation of ESCs in LIF-Free Culture Condition
Pluripotent ESCs harbor the ability to form EB. The knockdown of either Nme6 or Nme7 almost completely abolishes the ability of EB formation (Fig. 6A). Thus, Nme6 and Nme7 are essential for the EB formation. As mentioned previously, LIF is an important cytokine that can sustain ESC self-renewal. To clarify whether Nme6 or Nme7 supports ESC pluripotency in LIF-independent condition, we cultured Nme6- or Nme7-overexpressing ESCs in the culture medium with or without LIF for 5 days. Induction of Nme6 or Nme7 expression can rescue the EB formation ability for the ESCs cultured in the absence of LIF (Fig. 6B). The result suggested that Nme6 or Nme7 may be able to rescue LIF pathway and modulate the EB formation.
Nme6 and Nme7 Are Important for the Oncogenicity of ESCs
To elucidate the functions of Nme6 and Nme7 in vivo, teratoma formation assay was performed. The teratomas derived from the cells transfected with either shRNAs of Nme6 or Nme7 were significantly smaller than those derived from shLuc-transfected cells (Supporting Information Fig. S6A). The results indicated that Nme6 and Nme7 affect the oncogenicity of ESCs. In the teratoma, all tumors include the cells from ectoderm, endoderm, and mesoderm (Supporting Information Fig. S6B). This result suggested that the lineage differentiation may not be severely altered by the shNme6s or shNme7s in vivo, even though they affect the oncogenicity of ESCs.
Nme6 and Nme7 Regulates Oct4 Promoter
Because Oct4 is the master regulator and its transcriptional level is fast in response to Nme6 or Nme7 downregulation, we would like to investigate the mechanism of how Nme regulates the Oct4 expression. We therefore examined the subcellular localization of Nme6 and Nme7 in mouse ESCs. As shown in Figure 7A, Nme6 localizes at the cytoplasm while Nme7 both have nuclear and cytoplasmic localization. To further study the interplay of Oct4, Nme6, and Nme7, we performed luciferase assay with a luciferase reporter plasmid driven by the upstream promoter region of Oct4 (pGL3-Oct4). We verified that in Nme6 or Nme7 knockdown ESCs, there was a 50.2% or 63.1% decrease of activity compared to the controls (Fig. 7B). To further examine whether the overexpressed Nme6 or Nme7 can upregulate the Oct4 promoter activity, we decided to perform the luciferase assay in 293T cells to minimize background noise of luciferase signals by feed-forward circuit of Oct4. Our results demonstrated that ectopic expression of Nme6, Nme7v1, or Nme7v2 activates the Oct4 upstream promoter region, resulting in a 1.5-, 2.7-, and 3.7-fold increase in luciferase activity compared to vector control (Fig. 7C). Altogether, our results from luciferase assays provided evidence that Oct4 is a downstream target Nme6 and Nme7.
ESCs are characterized by its immortalization ability, pluripotency, and oncogenicity. However, the underlying mechanism is not fully revealed. We performed systematic functional screen in ESCs with 4,801 shRNA against 929 kinases and phosphatases. One hundred and thirty-two candidate genes that regulate both ESC expansion and stem cell marker expression are discovered (Supporting Information Table S2). Knockdown of each of the selected 27 genes induced morphological changes that are the signs of cell differentiation (Fig. 1). Among them, Nme6 and Nme7 were proven to be essential for ESC renewal (Figs. 2, 3, 5), EB formation (Fig. 6), and teratoma formation (Supporting Information Fig. S6). We further demonstrated that the overexpression of Nme6 or Nme7 can rescue the stem cell marker expression and the EB formation in the absence of LIF (Figs. 4, 6). This implies the importance of Nme6 and Nme7 in ESC renewal.
Functional screen with siRNA or shRNA becomes a powerful tool to identify multiple targets in ESC renewal. We performed a functional screen with ALP as the stem cell marker, because it is a well-accepted stem cell marker and also is a sensitive marker that shows up first in the iPSC reprogramming assay . It is more sensitive than the Oct4 expression and Oct4 reporter assay in the LIF removal testing (Supporting Information Fig. S1 and data not shown). Genome-wide siRNA screen had been previously performed in mouse ESCs with the pooled screening method followed by flow cytometry [24, 25]. In those previous studies, researchers used Oct4-GFP as the reporter system and robustly defined many Oct4 modulators. One group identified 296 hits in the primary screen while another group identified 148 hits. However, the results were not very consistent between these two laboratories, because only nine overlapping hits were identified. This might be because different designed siRNA libraries may have different sensitivity against each gene and lead to different results. In our screen, two overlapping hits has been identified in the previous study. In the screen, positive control is the shRNA against Oct4 that block ESC replication and induce differentiation. In our results, the shRNAs of 132 genes performed as the Oct4 shRNA in that they all reduce the expression of stem cell markers and cell number. The decrease of proliferation rate is one of the indicators of cell differentiation. In attempts to search for critical stemness factors, we launch a secondary screen with the morphological changes as the indicator, and then identified at least 27 candidate genes that participate in stem cell renewal. Interestingly, we found 130 candidate genes that regulate the cell number only and do not affect the activity of ALP (data not shown). It suggests that the decrease in cell number is not sufficient to induce cell differentiation or some other stem cell markers may have been affected despite that ALP activity is unchanged. Our screen is the first assay that can provide information of genes affecting ESC expansion.
The results from this shRNA screen revealed Nme6 and Nme7 significantly affect the expression of stem cell marker. Both Nme6 and Nme7 belong to the Nme family. The Nme family, previously known as Nm23 or NDPK, is involved in various molecular processes including promote or inhibit tumor metastasis depending on the cancer type . The inhibitor of a group I NDPK protein Nme2 can promote ESC differentiation . Although Nme6 and Nme7 belong to group II of NDPKs, Nme7 has NDPK domain but lacks NDPK activity . Both Nme6 and Nme7 are detected in colon cancer and gastric cancer but their functions remain unknown . Ectopic expression of Nme6 is detected in the mitochondria . Nme6 expresses abundantly in the early embryo in zebra fish . The expression of Nme6 is high in the oocyte and reaches the peak after zygotic genome activation. Nme6 then decreases after early embryogenesis. Nme7 abundantly expresses in human testis, ovary, or brain . Nme7 was also identified in the screens of proteasome antagonist for the inhibition of multiple myeloma proliferation . Nme7 is involved in ciliary transport of the hedgehog signaling . Nme7 may regulate vesicular transport of Golgi and acts as a component of the γ-tubulin ring complex [38, 39]. These research indicate the importance of Nme in development. The function of Nme7 in the nucleus remains unknown. Since Nme7 can regulate Oct4 promoter activity (Fig. 7), it will be of interest to investigate the functional roles of Nme7 in the nucleus.
Herein, we describe the identification of two novel stem cell regulators—Nme6 and Nme7—that can significantly increase the pluripotency of ESC by simultaneously modulating the expression of seven known critical factors for ESC renewal, including Oct4, Klf4, Dnmt3B, Nanog, ERas, Tert, and c-Myc. These results collectively demonstrate for the first time a role of Nme family proteins to be signaling hubs in ESC pluripotency. To our knowledge, it is the only factor that affects self-renewal in conjunction with most factors. Oct4, Klf4, Nanog, Sox2, and c-Myc are transcription factors to keep ESC in the pluripotent state. Tert and Dnmt3B work at DNA level to affect gene expression and telomere elongation. It is of note that among the Nme6 and Nme7 regulated genes, Oct4, Nanog, c-Myc, ERas, telomerase, and KLF4 all modulate tumorigenesis [14, 40-46]. Interestingly, Nme6 and Nme7 all four genes (Oct4, KLF4, c-Myc, and Sox2) that are sufficient for reprogramming iPSCs [16, 47, 48]. Nme6 and Nme7 also regulate Oct4 expression, the master regulator gene of ESCs.
The detailed mechanisms of how Nme6 or Nme7 regulates the pluripotency remain to be investigated. Based on the time course analysis, changes in Nme6 or Nme7 expression immediately provoke changes in expression of Oct4, KLF4, and c-Myc mRNA and the influence is faster than other ESC renewal genes. Therefore, Oct4, Klf4, and c-Myc might be more direct targets of Nme6 or Nme7, whereas Tert, Dnmt3B (response lately), and ERas (expression level is dynamic) might be indirect target given its slow response to the changes (Supporting Information Fig. S5). Nme6 or Nme7 probably regulated Tert expression through c-Myc . Up-to-date, several factors that modulate the expression of Oct4, Myc, and Tert in ESCs have been revealed [24–26], but the upstream regulators of KLF4, ERas, and Dnmt3B have not yet been defined. In this study, we also found Nanog/Sox2 was regulated by Nme6 or Nme7 at the post-transcriptional level (Fig. 2D), which suggests the versatile roles of Nme6 or Nme7 at the regulation of multiple key factors of ESCs.
Compared to undifferentiated ESC, the protein expression levels of Nme6 and Nme7 were downregulated upon EB formation (Fig. 2E). However, the expression level of Nme6 and Nme7 did not completely abolish after the differentiation. Several hypotheses may explain this phenomenon. First, it may suggest that the abundance expression of Nme6 or Nme7 might lead to the difference in the amplitude of the signals in different cell types. Alternatively, the activity of Nme6 or Nme7 may regulate at the post-translational level. Third, like LIF, PI3K, Wnt, or TGF-β signals, it is possible that Nme6 or Nme7 may have different roles in different cells [2, 22, 50-55].
The knockdown of either Nme6 or Nme7 diminishes the ability of EB formation, while the overexpression of each Nme6 or Nme7 can rescue the EB formation in the absence of LIF. This may act through the modulation of the key regulators such as Oct4, c-Myc, and Nanog or the participation of mediators of cell-cell interaction. Similarly, the teratoma formation is hampered in the presence of shNme6s and shNme7s (Supporting Information Fig. S6). The tumor weight is significantly less than the shLuc control (Supporting Information Fig. S6). This is consistent with the finding that Oct4 and ERas are important for teratoma formation [14, 56], and our finding that Nme6 or Nme7 both can regulate Oct4 and ERas. In addition, the knockdown of either Nme6 or Nme7 alters the expression of differentiation markers and promotes cell differentiation in vitro. It suggests that part of the cells might be differentiated and may loss the proliferating ability, which further results in less oncogenicity of ESCs. Thus, the Nme6 and Nme7 exert the effects largely on the expansion of ESCs, the formation of EB, and the generation of teratoma.
In light of the fact that ESC harbors tumorigenicity, characterization of ESC signaling pathway is thus believed to help the understanding of underlying mechanism of cancer. Both Nme6 and Nme7 are detected in colon cancer and gastric cancer but their functions remain unknown . ERas has been reported as an oncogene without any upstream inducers been identified . Nme6 and Nme7 are the first defined upstream signals of ERas. Oct4 and Nanog also express in undifferentiated tumors or cancer stem cells [42–46]. Extended from our results gained from ESCs, we hypothesized that Nme6 or Nme7 may be responsible for the regulation of ERas/Oct4/Nanog in cancers. It is of great interest to see the involvement of these stem cell factors in cancers to know whether Nme6 and Nme7 are oncogenes. If so, inhibition of Nme expression should hold promise for the future treatment. From this study, we identified Nme6 or Nme7 regulate multiple critical factors including Oct4, Nanog, Klf4, Myc, Tert, ERas, Sox2 and Dnmt3B. It increases our understanding in stem cell biology.
From the shRNA screen, 132 novel genes essential for ESC renewal were identified. 27 of them might be key regulators of ESCs since downregulation of these genes efficiently induced ESC differentiation. These genes were novel and were not identified by other shRNA screens. Among the candidate genes, Nme6 and Nme7, are found to be essential for the expression of multiple stem cell key regulators including Oct4, Nanog, Klf4, c-Myc, telomerase, Dnmt3B, Sox2 and ERas. Either knockdown of Nme6 or Nme7 induce the expression of differentiation markers and reduces the formation of EB and teratoma. The overexpression of Nme6 or Nme7 rescued the expression of multiple stem cell core regulators and EB formation. This finding identified multiple novel candidates for ESC renewal and reveals the critical roles of Nme6 and Nme7 in ESC expansion, pluripotency, and oncogenicity.
This work was supported by the National Health Research Institutes (NHRI-EX100-10025SI), National Science Council (NSC-100-2314-B-001-002 NSC-97-2321-B-001-022-MY3, and NSC-100-2321-B-001-039-), summit project, and Academia Sinica. Nianhan Ma is supported by 5500 grant, NCU, Aim for Top University Plan. We thank Dr. Ming-Ji Fann and Dr. Ching Hwa Tsai for their suggestions in this manuscript. We also thank I-Huan Liao, Chih-Han Chien, Shang-Chih Yang, National RNAi Core facility, and the fluorescence microscope core facility for their technical assistance. We thank the Taiwan Mouse Clinic that is funded by the National Research Program for Biopharmaceuticals (NRPB) at the NSC of Taiwan for technical support in the analysis of histology data.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.