**3. Oncogenes that are involved in ES cell self-renewal**

#### **3.1. β-catenin**

STAT3 is constitutively activated in nearly 70% of tumors. Because of the high frequency of its

Klfs are a family of transcription factors that play important roles in many fundamental biological processes. They were named "Krüppel-like" owing to their strong homology with the *Drosophila* gene product Krüppel, which is involved in segmentation of the develop‐ ing embryo. Klf family proteins contain three C2H2-type zinc fingers that bind DNA. *Klf4* was independently cloned by two groups and named "gut-enriched KLF" and "epithelial zinc finger" owing to its high expression in the intestine and skin epithelium, respective‐ ly [59, 60]. However, it was later discovered that this transcription factor is expressed in several other tissues, such as lung, testis, and thymus. In addition to a C-terminal DNAbinding domain consisting of zinc fingers, Klf4 protein contains an activation domain in its N-terminus and a repressive domain in its central region. Probably owing to this struc‐ ture, Klf4 is a bi-functional transcription factor that can either activate or repress transcrip‐ tion of its target genes. Similar to Oct3/4 and Sox2, Klf4 is one of the four factors that induce

Klf4 is highly expressed in self-renewing ES cells, but not in differentiated ES cells. Klf4 regulates self-renewal-specific expression of Lefty1, in cooperation with Oct3/4 and Sox2 [61]. Klf4 is also involved in Oct3/4 expression [62]. Overexpression of Klf4 results in the inhibition of ES cell differentiation, possibly through upregulation of *Nanog* [63, 64]. In addition, there is a marked overlap between genes that are regulated by Nanog and those that are regulated by Klf4. These observations suggest the importance of Klf4 in ES cell self-renewal. However, *Klf4*-null mice have no detectable defects during embryogenesis [65]. Furthermore, a recent study reported that the function of Klf4 in ES cell self-renewal is partially redundant because combined knockdown of *Klf4*, *Klf2*, and *Klf5*, but not any one gene individually, results in spontaneous ES cell differentiation [62], suggesting that Klf4 is dispensable for ES cell self-

In cancer cells, Klf4 acts as a tumor suppressor or an oncogene, possibly owing to its bifunctionality. Whether Klf4 acts as a tumor suppressor or an oncogene likely depends on the tumor type. For example, Klf4 functions as a tumor suppressor in the intestinal and gastric epithelium, and expression of *Klf4* is downregulated in human colorectal and gastric carcino‐ mas [66, 67]. On the other hand, overexpression of Klf4 in the skin results in squamous epithelial dysplasia, eventually leading to squamous cell carcinoma [68, 69]. A high expression level of Klf4 significantly correlates with a poor prognosis in hepatocellular carcinoma [70].

Zfp57 is a transcription factor that was originally identified as an undifferentiated cell-specific gene in F9 embryonal carcinoma cells [71]. Mouse Zfp57 protein contains one Kruppelassociated box (KRAB) domain and five zinc fingers, while human Zfp57 protein has one KRAB domain and seven zinc fingers. In adult mouse, Zfp57 is highly expressed in testis and

overactivation, STAT3 is considered to be a valuable target for anti-cancer therapy.

46 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

**2.5. Krüppel-like factor (Klf) 4**

reprogramming of murine cells [19].

**2.6. Zinc-finger protein (Zfp)-57**

renewal.

The importance of the Wnt pathway in tumorigenesis was recognized by identification of adenomatous polyposis coli (APC) mutations in familial adenomatous polyposis [78, 79]. In Wnt signaling, the Apc protein functions as a negative regulator and is involved in degradation of β-catenin, the mammalian homologue of *Drosophila* Armadillo. β-catenin contains multiple armadillo repeats in its central region and a transcriptional activator domain in its C-terminal region. Human and mouse β-catenin proteins both have 781 amino acid residues and are almost identical to each other (only one amino acid difference). β-catenin acts as a transcrip‐ tional co-activator and an adaptor protein for intracellular adhesion. In epithelial tissues, βcatenin interacts with cadherins and α-catenin, and regulates epithelial cell growth and intracellular adhesion. By contrast, in Wnt signaling, β-catenin is a major transcriptional modulator and plays a crucial role in embryogenesis. In the absence of Wnt signaling, Apc forms a complex with β-catenin and Axin. This leads to phosphorylation of β-catenin by glycogen synthase kinase (GSK)-3β, which triggers degradation of β-catenin. When Wnt binds to its receptor Frizzled, Disheveled is hyper-phosphorylated, which results in release of GSK3β from the β-catenin degradation complex and prevents phosphorylation of β-catenin by GSK3β. Unphosphorylated β-catenin translocates into the nucleus, where it forms a complex with Tcf/Lef and functions as a transcriptional co-activator.

as their expression of stemness genes, including *Oct3/4* and *Sox2* [90]. Gli1 forms a positive feedback loop with Nanog, and the Nanog/Gli1 signaling axis is indispensable for regulation of glioma stem cells [91]. In Ewing sarcoma, expression of Gli1 is regulated in an Hh-inde‐ pendent manner: EWS-FLI, an oncogenic transcription factor that is produced by chromosomal

Embryonic Stem Cells and Oncogenes http://dx.doi.org/10.5772/57614 49

Gli1 and Gli2 are both highly expressed in undifferentiated ES cells, while Gli3 expression level is low [94]. Gli1 and Gli2 are downstream molecules of Oct3/4 and Nanog, and their expression is downregulated upon differentiation. When Gli1 and Gli2 are suppressed by a dominantnegative mutant of Gli2, expression of the self-renewal marker Sox2 decreases, whereas that of the differentiation markers Gata4 and Cdx2 increases, suggesting the importance of Gli activity for ES cell self-renewal. However, expression of this dominant-negative Gli2 mutant does not affect expression of the self-renewal markers Oct3/4 and Nanog. These findings suggest that Gli activity is involved in repressing ES cell differentiation, but is dispensable for

The serine/threonine protein kinase Akt was independently identified by three different groups. Two groups identified this kinase as being homologous to protein kinase C and protein kinase A, giving rise to the names "protein kinase B" and "RAC-PK" (related to the A and C kinases) [96, 97]. The other group identified this kinase as the cellular counterpart of the oncogene *v-akt* of the acutely transforming retrovirus AKT8 that is found in a rodent T-cell lymphoma [98]. Here, I will describe this protein as "Akt". Akt has three isoforms, namely, Akt1, Akt2, and Akt3. Each Akt family member has an N-terminal pleckstrin homology domain, a short α-helical linker, and a C-terminal kinase domain. Akt is directly downstream of phosphatidylinositol-3-OH kinase, and is a key player in the regulation of cell growth and

As expected from its identification as a counterpart of a viral oncogene, Akt plays an important role in human malignancy [99, 100]. Several studies have identified amplification of the *Akt* gene in human cancers. Amplification of *Akt1* was detected in a human gastric cancer [101]. Amplification and overexpression of Akt2 were detected in ovarian and pancreatic cancers. Artificial activation of Akt1 or Akt2 can transform NIH3T3 cells [102, 103], and Akt2 anti-sense RNA inhibits the tumorigenic phenotype of pancreatic carcinoma cell lines [104]. Furthermore, Akt1 kinase activity is often increased in prostate and breast cancers and is associated with a

A constitutively activated Akt mutant can maintain the undifferentiated phenotype of mouse ES cells, even in the absence of LIF, although the mechanism underlying Akt-mediated maintenance of ES cell self-renewal is unclear [105]. Bechard and Dalton demonstrated that Akt phosphorylates, and thereby inactivates, GSK3β in ES cells, suggesting that Akt maintains ES cell self-renewal by inactivating GSK3β and thus stimulating activation of β-catenin [106]. By contrast, Watanabe *et al.* did not observe the accumulation of β-catenin in nuclei or activation of the transcriptional activity of β-catenin in mouse ES cells [105]. Another possible

translocation, directly upregulates *Gli1* expression to promote tumor growth [92, 93].

ES cell self-renewal. In addition, Gli is involved in ES cell growth [94, 95].

**3.3. Akt**

survival.

poor prognosis [102].

In human cancers, such as colon cancer, the β-catenin/Tcf/Lef complex positively regulates the expression of a variety of cancer-associated genes, including *cyclin D*, *Tert*, and *c-Myc*, to promote tumorigenesis. Apc mutation leads to stabilization and accumulation of β-catenin in nuclei. However, in some cancers, mutation of β-catenin itself renders this protein unable to be phosphorylated, resulting in its stabilization.

Accumulated evidence suggests that Wnt/β-catenin signaling contributes to the maintenance of ES cell self-renewal. For example, *Apc*-null ES cells show severe differentiation defects [80]. Undifferentiated ES cells can be maintained in a self-renewing state by using conditioned medium from Wnt3a-expressing cells [81]. Furthermore, enforced expression of an activated form of β-catenin maintains the self-renewal of ES cells, even in the absence of LIF [23, 82]. Expression of γ-catenin, which has a similar structure to β-catenin, partially sustains the selfrenewal of ES cells in the absence of LIF [23]. β-catenin binds to Oct3/4 to enhance its tran‐ scriptional activity in ES cells, leading to upregulation of *Nanog*, a target gene of Oct3/4 [23]. The Wnt/β-catenin pathway upregulates expression of *STAT3*, and this signaling converges with that of LIF [83]. Similarly, Wnt and LIF work in synergy to maintain the pluripotency of mouse ES cells [84]. On the other hand, the self-renewal of ES cells can be maintained without β-catenin [85]. Taken together, these results suggest that β-catenin promotes, but is dispensa‐ ble, for ES cell self-renewal. Moreover, it was recently shown that ES cells lacking Wnt signaling resemble epiblast stem cells in terms of their morphology and gene expression [86, 87]. This suggests that Wnt/β-catenin signaling prevents the transition of ES cells from a naïve to a primed pluripotent state. In addition, β-catenin regulates Tert expression in ES cells [88], as is the case in cancer cells.

#### **3.2. Gli**

The zinc-finger transcription factor Gli is a central player in the Hedgehog (Hh)-mediated signaling pathway, which plays a critical role during embryogenesis. Gli belongs to the Klf family and has three isoforms in mammals, namely, Gli1, Gli2, and Gli3. Gli1 and Gli2 usually act as transcription activators, while Gli3 is a transcription suppressor. All Gli proteins have a DNA-binding domain consisting of C2-H2 class zinc fingers. In addition, Gli1 and Gli2 contain a C-terminal transactivation domain, while Gli2 and Gli3 have an N-terminal repres‐ sion domain. Hh family proteins, namely, Sonic Hh, Indian Hh, and Desert Hh, function as ligands of the transmembrane receptor Ptch1. In the absence of a Hh ligand, Ptch1 inhibits the activity of the G-protein coupled receptor-like protein Smo, resulting in formation of a complex of Gli2 and Gli3 with the inhibitory protein Sufu. This results in cleavage of Gli2 and Gli3 into their repressor forms, which translocate into the nucleus. By contrast, binding of Hh ligands to Ptch1 results in the release and activation of Smo, leading to activation and nuclear trans‐ location of Gli2, which results in transactivation of target genes, including *Gli1*.

*Gli1* was originally identified as an amplified gene in a human glioma cell line [89]. It was recently shown that Hh/Gli signaling regulates the self-renewal of glioma stem cells, as well as their expression of stemness genes, including *Oct3/4* and *Sox2* [90]. Gli1 forms a positive feedback loop with Nanog, and the Nanog/Gli1 signaling axis is indispensable for regulation of glioma stem cells [91]. In Ewing sarcoma, expression of Gli1 is regulated in an Hh-inde‐ pendent manner: EWS-FLI, an oncogenic transcription factor that is produced by chromosomal translocation, directly upregulates *Gli1* expression to promote tumor growth [92, 93].

Gli1 and Gli2 are both highly expressed in undifferentiated ES cells, while Gli3 expression level is low [94]. Gli1 and Gli2 are downstream molecules of Oct3/4 and Nanog, and their expression is downregulated upon differentiation. When Gli1 and Gli2 are suppressed by a dominantnegative mutant of Gli2, expression of the self-renewal marker Sox2 decreases, whereas that of the differentiation markers Gata4 and Cdx2 increases, suggesting the importance of Gli activity for ES cell self-renewal. However, expression of this dominant-negative Gli2 mutant does not affect expression of the self-renewal markers Oct3/4 and Nanog. These findings suggest that Gli activity is involved in repressing ES cell differentiation, but is dispensable for ES cell self-renewal. In addition, Gli is involved in ES cell growth [94, 95].

#### **3.3. Akt**

GSK3β from the β-catenin degradation complex and prevents phosphorylation of β-catenin by GSK3β. Unphosphorylated β-catenin translocates into the nucleus, where it forms a

In human cancers, such as colon cancer, the β-catenin/Tcf/Lef complex positively regulates the expression of a variety of cancer-associated genes, including *cyclin D*, *Tert*, and *c-Myc*, to promote tumorigenesis. Apc mutation leads to stabilization and accumulation of β-catenin in nuclei. However, in some cancers, mutation of β-catenin itself renders this protein unable to

Accumulated evidence suggests that Wnt/β-catenin signaling contributes to the maintenance of ES cell self-renewal. For example, *Apc*-null ES cells show severe differentiation defects [80]. Undifferentiated ES cells can be maintained in a self-renewing state by using conditioned medium from Wnt3a-expressing cells [81]. Furthermore, enforced expression of an activated form of β-catenin maintains the self-renewal of ES cells, even in the absence of LIF [23, 82]. Expression of γ-catenin, which has a similar structure to β-catenin, partially sustains the selfrenewal of ES cells in the absence of LIF [23]. β-catenin binds to Oct3/4 to enhance its tran‐ scriptional activity in ES cells, leading to upregulation of *Nanog*, a target gene of Oct3/4 [23]. The Wnt/β-catenin pathway upregulates expression of *STAT3*, and this signaling converges with that of LIF [83]. Similarly, Wnt and LIF work in synergy to maintain the pluripotency of mouse ES cells [84]. On the other hand, the self-renewal of ES cells can be maintained without β-catenin [85]. Taken together, these results suggest that β-catenin promotes, but is dispensa‐ ble, for ES cell self-renewal. Moreover, it was recently shown that ES cells lacking Wnt signaling resemble epiblast stem cells in terms of their morphology and gene expression [86, 87]. This suggests that Wnt/β-catenin signaling prevents the transition of ES cells from a naïve to a primed pluripotent state. In addition, β-catenin regulates Tert expression in ES cells [88], as is

The zinc-finger transcription factor Gli is a central player in the Hedgehog (Hh)-mediated signaling pathway, which plays a critical role during embryogenesis. Gli belongs to the Klf family and has three isoforms in mammals, namely, Gli1, Gli2, and Gli3. Gli1 and Gli2 usually act as transcription activators, while Gli3 is a transcription suppressor. All Gli proteins have a DNA-binding domain consisting of C2-H2 class zinc fingers. In addition, Gli1 and Gli2 contain a C-terminal transactivation domain, while Gli2 and Gli3 have an N-terminal repres‐ sion domain. Hh family proteins, namely, Sonic Hh, Indian Hh, and Desert Hh, function as ligands of the transmembrane receptor Ptch1. In the absence of a Hh ligand, Ptch1 inhibits the activity of the G-protein coupled receptor-like protein Smo, resulting in formation of a complex of Gli2 and Gli3 with the inhibitory protein Sufu. This results in cleavage of Gli2 and Gli3 into their repressor forms, which translocate into the nucleus. By contrast, binding of Hh ligands to Ptch1 results in the release and activation of Smo, leading to activation and nuclear trans‐

location of Gli2, which results in transactivation of target genes, including *Gli1*.

*Gli1* was originally identified as an amplified gene in a human glioma cell line [89]. It was recently shown that Hh/Gli signaling regulates the self-renewal of glioma stem cells, as well

complex with Tcf/Lef and functions as a transcriptional co-activator.

48 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

be phosphorylated, resulting in its stabilization.

the case in cancer cells.

**3.2. Gli**

The serine/threonine protein kinase Akt was independently identified by three different groups. Two groups identified this kinase as being homologous to protein kinase C and protein kinase A, giving rise to the names "protein kinase B" and "RAC-PK" (related to the A and C kinases) [96, 97]. The other group identified this kinase as the cellular counterpart of the oncogene *v-akt* of the acutely transforming retrovirus AKT8 that is found in a rodent T-cell lymphoma [98]. Here, I will describe this protein as "Akt". Akt has three isoforms, namely, Akt1, Akt2, and Akt3. Each Akt family member has an N-terminal pleckstrin homology domain, a short α-helical linker, and a C-terminal kinase domain. Akt is directly downstream of phosphatidylinositol-3-OH kinase, and is a key player in the regulation of cell growth and survival.

As expected from its identification as a counterpart of a viral oncogene, Akt plays an important role in human malignancy [99, 100]. Several studies have identified amplification of the *Akt* gene in human cancers. Amplification of *Akt1* was detected in a human gastric cancer [101]. Amplification and overexpression of Akt2 were detected in ovarian and pancreatic cancers. Artificial activation of Akt1 or Akt2 can transform NIH3T3 cells [102, 103], and Akt2 anti-sense RNA inhibits the tumorigenic phenotype of pancreatic carcinoma cell lines [104]. Furthermore, Akt1 kinase activity is often increased in prostate and breast cancers and is associated with a poor prognosis [102].

A constitutively activated Akt mutant can maintain the undifferentiated phenotype of mouse ES cells, even in the absence of LIF, although the mechanism underlying Akt-mediated maintenance of ES cell self-renewal is unclear [105]. Bechard and Dalton demonstrated that Akt phosphorylates, and thereby inactivates, GSK3β in ES cells, suggesting that Akt maintains ES cell self-renewal by inactivating GSK3β and thus stimulating activation of β-catenin [106]. By contrast, Watanabe *et al.* did not observe the accumulation of β-catenin in nuclei or activation of the transcriptional activity of β-catenin in mouse ES cells [105]. Another possible mechanism is that Akt induces expression of Tbx3, which in turn stimulates expression of Nanog to maintain ES cell self-renewal [107].

Since ES cells and iPS cells have similar gene expression profiles, it is likely that these common transcription factors are also expressed in iPS cells. Considering the transforming potential of these factors, it is possible that their high expression in iPS cells increases the risk of tumor formation during cell therapy using iPS-derived cells. Indeed, we have already found that

Embryonic Stem Cells and Oncogenes http://dx.doi.org/10.5772/57614 51

In this way, understanding the roles of putative oncogenes in ES cells will not only help to elucidate the molecular basis underlying the similarity between ES cells and cancers cells, but will also help to develop a novel method that can be used in cancer therapy and regenerative

**Figure 2.** Transcription factor network that regulates ES cell self-renewal. Considering the similarities between ES cells

Department of Stem Cell Biology, Graduate School of Medical Sciences, Kanazawa University,

and cancer cells, at least a part of this network may be used for growth regulation in cancer cells.

Address all correspondence to: hkoide@med.kanazawa-u.ac.jp

*Zfp57*-null ES cells are significantly less able to form tumors than wild-type ES cells.

medicine.

**Author details**

Hiroshi Koide\*

Kanazawa, Japan

#### **3.4. c-Myc**

c-Myc is a cellular counterpart of the *v-myc* gene, which was isolated from the avian retrovirus MC29 [108], and belongs to a family of helix-loop-helix/leucine zipper transcription factors. c-Myc forms a complex with Max, which results in the increased stability of c-Myc protein [109]. Phosphorylation of Thr-62 by Erk also stabilizes c-Myc, while phosphorylation of Thr-58 by GSK3β reduces the stability of c-Myc [110]. c-Myc regulates expression of its target genes through binding to E-box sequences and recruiting histone acetyltransferases. Under normal conditions, when cells are stimulated by an internal or external growth-promoting signal, the level of c-Myc rapidly and transiently increases to induce cell proliferation, and the level of c-Myc subsequently returns to a low level in quiescent cells.

It is well-established that many, if not most, human tumors have elevated levels of c-Myc owing to gene amplification and translocation [111, 112]. Gene amplification of *c-Myc* has been reported in several cancers, including breast, ovarian, and colon. A common human translo‐ cation involving *c-Myc* is t(8;14), which is critical for the development of Burkitt's lymphoma. Although c-Myc was one of the four factors originally identified as being required for iPS cell production [19], generation of iPS cells without this transcription factor has been reported [113], suggesting that c-Myc is dispensable for cell reprogramming.

During the self-renewal of ES cells, levels of c-Myc are elevated [114]. By contrast, upon LIF withdrawal, the level of *c-Myc* mRNA decreases and c-Myc protein is phosphorylated on Thr-58 by GSK3β, which triggers degradation of c-Myc. Expression of a stable c-Myc mutant, in which the Thr-58 residue is mutated to alanine, allows ES cells to self-renew in the absence of LIF. By contrast, expression of a dominant-negative form of c-Myc inhibits the self-renewal of ES cells and induces their differentiation. These findings suggest that c-Myc is critically involved in maintaining the self-renewal of ES cells. Moreover, analysis of *Max*-null ES cells revealed that the function of c-Myc/Max in ES cell self-renewal seems to be largely independent of the Oct3/4, Sox2, and Nanog regulatory networks [115].
