Mouse ES (embryonic stem) cells maintain pluripotency with robust proliferation in vitro. ES cells share some similarities with cancer cells, such as anchorage-independent growth, loss of contact inhibition and tumour formation. After differentiation, ES cells lose pluripotency and tumorigenicity. Recent studies showed that the PI3K (phosphoinositide 3-kinase) pathway is important for proliferation, survival and maintenance of pluripotency in ES cells. The PI3K pathway is activated by growth factors and cytokines including insulin and leukaemia inhibitory factor. In addition to these exogenous factors, the PI3K pathway is endogenously activated by the constitutively active Ras family protein ERas (ES cell-expressed Ras). The PI3K pathway utilizes multiple downstream effectors including mTOR (mammalian target of rapamycin), which we have shown to be essential for proliferation in mouse ES cells and early embryos.
- embryonic stem cell-expressed Ras (ERas)
- mammalian target of rapamycin (mTOR)
- phosphoinositide 3-kinase (PI3K)
Embryonic stem (ES) cells were first established from the inner cell mass of mouse blastocysts in 1981 [1,2]. ES cells can proliferate infinitely while maintaining pluripotency, the ability to differentiate into any kind of cells existing within the animal body. Pluripotent cells were subsequently generated from human blastocysts in 1998, which are considered to be promising sources for cell therapy to treat patients with degenerative diseases such as diabetes and Parkinson's disease . However, ES cells have a propensity to produce tumours called teratoma when transplanted, which may preclude their therapeutic usage.
Transcription factors expressed predominantly in ES cells, such as Oct3/Oct4 [4,5] and Nanog [6,7], have been shown to be essential for the maintenance of pluripotency. Several cytokines including LIF (leukaemia inhibitory factor), BMP4 (bone morphogenetic protein 4) and Wnt could sustain self-renewal of ES cells [8–11]. However, it remains elusive how ES cells possess tumour-like properties without having chromosomal abnormalities.
In this review, we introduce the role of PI3K (phosphoinositide 3-kinase) in tumour-like properties and self-renewal in ES cells.
Tumorigenicity and growth properties of ES cells
ES cells share many growth properties with tumour cells. Both ES cells and tumour cells can grow infinitely without replicative senescence when cultured under optimal conditions. Anchorage-independent growth, losing contact inhibition and tumorigenicity in vivo are also in common. When ES cells are subcutaneously injected into immunodeficient or isogenic mice, a teratoma is formed within a few weeks. This tumour is composed of all three germ layers in a disorganized fashion, and similar tumours have been found in testicular carcinoma. Particularly, strain 129 background mice have a high incidence of teratocarcinoma formation, and ES cells can be established from 129-strain much more easily than from other strains. Thus there could be some common molecular mechanisms shared by ES cells and tumour cells.
ES cells were first recognized as EC (embryonic carcinoma)-like cells [1,2]. These cells can propagate robustly and infinitely in the presence of serum and LIF [8,10]. In addition, co-culture with mouse embryonic fibroblasts also supports ES cell self-renewal. Under these conditions, murine ES cells can propagate from a single cell. Their growth properties allow us to obtain monoclonal mutants by gene targeting and therapeutic sources. EC cells are derived from teratocarcinomas and contain chromosomal abnormalities such as aneuploid. In contrast, ES cells are established from the inner cell mass of the blastocyst, and have no genetic abnormalities. When ES cells are injected into blastocysts, they can contribute to all of the somatic and germ cells. How can ES cells possess tumour-like properties despite having no mutations in proto-oncogenes or tumour suppressor genes? The molecular mechanisms of tumorigenicity and rapid growth in undifferentiated ES cells are unclear.
Recent studies indicate the important role of PI3K in the tumour-like properties of ES cells. PI3Ks are enzymes that phosphorylate phospholipids at the plasma membrane. These enzymes consist of a p110 catalytic subunit and adaptor subunit complexes. Normally, PI3Ks are regulated and activated by growth factors via receptor tyrosine kinases. Activated PI3K phosphorylates PtdIns(3,4)P2 and generates PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 is a target of PH domain (pleckstrin homology domain)-containing proteins and acts as a second messenger. Proteins such as Akt interact with PtdIns(3,4,5)P3 via PH domains and are subsequently translocated to the plasma membrane. Activation of Akt plays important roles in cell proliferation and survival by phosphorylating various substrates. Thus PI3K activity mediates various signalling pathways from cytokines to downstream targets.
PTEN (phosphatase and tensin homologue deleted on chromosome ten)
PTEN is a tumour suppressor and acts as a negative regulator of the PI3K pathway by dephosphorylation of PtdIns(3,4,5)P3. Mutation or deletion of the PTEN gene has been identified in various human cancers such as brain, breast and prostate cancers . Deletion of the mouse PTEN gene revealed that PTEN is essential for early embryonic development [13–16]. PTEN-null embryos died by 9.5 days post coitum (dpc). In addition, survival rates of PTEN+/− mice were decreased.
Sun et al.  showed that PTEN-deficient ES cells grew much faster than wild-type ES cells. In addition, tumorigenicity of these cells was enhanced. Actually, PTEN knockout mice have a high incidence of testicular carcinoma . In PTEN-null ES cells, PI3K activity was markedly enhanced and its downstream targets such as Akt were activated. At the same time, cyclin D1 protein was increased in these cells. In addition, expression levels of the cyclin-dependent kinase inhibitor p27kip1 were decreased. These results suggest that PTEN negatively regulates ES cell proliferation in vitro and teratoma formation in vivo.
This group also showed that deletion of Akt1, also known as protein kinase Bα, by gene targeting in PTEN-null ES cells resulted in suppression of both tumorigenicity and proliferation rate in vitro . Akt1 and PTEN double-knockout ES cells proliferated much slower than wild-type ES cells. These results indicated that Akt functions as a downstream effector of PI3K in ES cell growth. However, ES cells deficient in PDK1 (phosphoinositide-dependent kinase 1) grew normally . Phosphorylation by PDK1 is required for the activation of Akt, and loss of Akt activity was observed in PDK1-deficient cells. Therefore the significance of Akt in ES cell proliferation remains unclear.
Roles of PI3K in cell-cycle control of ES cells were demonstrated by Jirmanova et al.  who showed that treatment with LY294002, which is a specific inhibitor of PI3K, markedly increased G0/G1 phase in ES cells . These results indicate that PI3K promote ES cell proliferation and tumorigenicity.
However, PI3K activity exists not only in undifferentiated ES cells but also in non-tumorigenic cells such as differentiated ES cells or somatic cells. One possibility is that the PI3K pathway is activated by unique mechanisms in undifferentiated ES cells. This hypothesis was demonstrated by the discovery of ERas (ES cell-expressed Ras).
By analysing the mouse EST (expressed sequence tag) databases with digital differential display, we have identified approx. 20 ES cell-specific genes [22–24]. These genes were termed ECATs (ES cell-associated transcripts).
One of these ECATs, ECAT5, encodes a polypeptide with approx. 40% identify with HRas, KRas and NRas, and was named ERas . The ERas gene is located on X-chromosome, and conserved in mouse, rat and human. ERas is expressed specifically in mouse ES cells but not in differentiated ES cells or somatic tissues derived from adult mice. In ERas proteins, the five domains that are essential for small G-proteins are highly conserved (Figure 1A). It also contains a CAAX motif and is located at the cytoplasmic membrane (Figure 1B).
ERas protein contains three amino acid substitutions at the positions that are important for GTPase activity of Ras proteins. Biochemical analysis showed that >95% of ERas protein existed in an activated form in ES cells. Assays with NIH3T3 cells demonstrated that ERas was an oncogenic protein because of its constitutive activation. Forced expression of ERas or HRasV12 (a constitutively active mutant of HRas) in NIH3T3 cells induced malignant transformation. When these cells were subcutaneously injected into mice, tumour formation was observed.
Forced expression of HRasV12 in PMEFs (primary mouse embryonic fibroblasts) causes premature senescence. These senescent cells become flattened and could no longer proliferate. In contrast, ERas does not induce premature senescence, but rather promotes PMEF proliferation. These results suggested that the biological functions of ERas differed from those of HRas.
Ras family proteins elicit their functions through binding to downstream effectors. HRas, KRas and NRas use Raf1 serine-threonine kinase, RalGDS (Ral guanine nucleotide dissociation stimulator) and PI3K as effectors to modify cell proliferation, apoptosis and differentiation. In ES cells, forced expression of HRasV12 induces massive differentiation through activation of the MAPK (mitogen-activated protein kinase) pathway (Figure 1C). In great contrast, ERas binds to PI3K but not to Raf1 or RalGDS. The phosphorylation status of Akt was significantly decreased in ERas-deficient ES cells. These results demonstrated that ERas specifically binds and activates PI3K as an effector.
Blastocyst microinjection of ERas-null ES cells produced germline-transmitted chimaeric mice. In addition, ERas-deficient mice developed normally and are fertile. This indicates that ERas is not essential for the maintenance of pluripotency and animal development.
ERas-deficient ES cells formed significantly smaller teratomas and had a reduced proliferation rate compared with wild-type ES cells. These phenotypes in ERas-deficient cells were rescued by re-expression of ERas or forced expression of activated PI3K cDNA. These results indicate that ERas and its downstream effector PI3K promote ES cell proliferation and tumorigenicity.
However, ERas-null ES cells still produce teratomas, albeit smaller than those produced by normal ES cells, suggesting the existence of other factors promoting tumorigenicity. Factors involved in other signalling pathways might also be involved in tumorigenicity of ES cells. Identification of these factors is essential for understanding the tumour-like properties of ES cells and for their clinical application.
mTOR (mammalian target of rapamycin)
mTOR, also known as FRAP (FKBP12-rapamycin-associated protein), RAFT (rapamycin and FKBP12 target), and RAPT (rapamycin target), belongs to the PIKK (phosphoinositide kinase-related kinase) family [25,26]. mTOR and other members of this family, including ATM (ataxia terangiectasis mutated), ATR (ataxia terangiectasia and Rad3-related) and DNA-PKcs (catalytic subunit of the DNA-dependent protein kinase), contain C-terminal regions with high indentity with the catalytic domain of PI3K . However, no PIKK members have lipid kinase activity. TOR protein phosphorylates serine or threonine residues . The mTOR gene encodes a 290 kDa serine/threonine kinase and is evolutionarily conserved from budding yeast to human. This large protein controls protein synthesis, in part by phosphorylating downstream substrates, including p70 S6 kinase [29,30] and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) . mTOR can be regulated by the PI3K pathway.
Recently, we showed that mTOR is essential for both ES cell proliferation and mouse development . Embryos carrying mTOR-null mutation died at 6.5 dpc. Histological analyses indicated that mTOR-deficient embryos showed impaired cell proliferation and gastrulation (Figure 2). Mutant blastocysts showed normal morphologies but could not propagate when cultivated in vitro. Conditional knockout of the mTOR gene by using Cre recombinase induced growth retardation in ES cells. In addition, treatment with rapamycin, which is a TOR inhibitor, interferes with ES cell proliferation. Thus mTOR is a strong candidate for a downstream target of the ERas/PI3K signalling in ES cells.
Recent studies have demonstrated that the tumour suppressors TSC1 (tuberous sclerosis complex 1) and TSC2 form a heterodimer that inhibits mTOR activities. In addition, the small GTPase Rheb (Ras homologue enriched in brain) was recently identified as a molecular target of TSCs . Rheb is a small GTPase protein and enhances activity of mTOR, but not that of Akt. TSC complex negatively regulates the activity of the mTOR pathway through the inactivation of Rheb. It has been shown that TSC2, which contains a C-terminal GTPase-activator domain, directly stimulates Rheb GTPase activity in vitro. How Rheb regulates mTOR function is as yet unclear . Rheb, TSC1 and TSC2 are expressed in undifferentiated ES cells. Thus the mTOR/TSC/Rheb pathway is likely to be an important downstream effector of PI3K that enhances ES cell proliferation and growth.
Role of PI3K in maintenance of the undifferentiated state of ES cells
In addition to proliferation and tumorigenicity, PI3K activity might be also crucial for self-renewal of ES cells (Figure 3). Paling et al.  reported that PI3K was important for maintenance of the undifferentiated state of ES cells by forced expression of dominant-negative mutant and treatment with LY294002, a specific inhibitor of PI3K. They proposed that PI3K may promote self-renewal by inhibiting the Ras/MAPK pathway, but precise mechanisms remain elusive.
Lin et al.  reported that tumour suppressor p53 promoted differentiation of ES cells by suppressing Nanog expression. They also showed that suppression of Nanog by p53 depended on the phosphorylation status of Ser315 of p53. It is known that this residue is a substrate of GSK3β (glycogen synthase kinase 3β) [37,38]. The important role of GSK3 in ES cell self-renewal was addressed by Sato et al. . They showed that BIO (6-bromoindirubin-3′-oxime), which is a GSK3-specific inhibitor derived from mollusc Tyrian Purple, supported the maintenance of human and mouse ES cells in an undifferentiated state. GSK3 is negatively regulated by PI3K and Akt . Taken together, PI3K may contribute to self-renewal of ES cells through GSK3β, p53 and Nanog.
Another pluripotent stem cell line, EG (embryonic germ) cells, can be established from PGCs (primordial germ cells). EG cells have many similarities to ES cells in growth properties, in differentiation potentials and in gene expression patterns . The derivation of EG cells from PGCs requires the supplementation of bFGF (basic fibroblast growth factor), stem cell factor and LIF. Noteworthily, all of these molecules can stimulate the PI3K pathway. Kimura et al.  performed conditional knockout of the PTEN gene in PGCs. Inactivation of PTEN increased PGC proliferation and the efficiency of EG cell derivation. In addition, PGC growth was promoted by forced expression of Akt and inhibited by a dominant-negative mutant of Akt. Therefore these results indicate that PI3K and Akt positively regulate PGC and EG cell proliferation .
We thank all the members of Yamanaka laboratory for useful discussions, encouragement and technical and administrative support.
Stem Cells and Development: A Focus Topic at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by T. Kouzarides (Cambridge, U.K.), S. Newbury (Newcastle upon Tyne, U.K.), B. Richardson (University College London, U.K.), R. Sablowski (John Innes Centre, Norwich, U.K.), D. Tosh (Bath, U.K.), M. Welham (Bath, U.K.) and A. Willis (Nottingham, U.K.).
Abbreviations: dpc, days post coitum; EC, embryonic carcinoma; EG, embryonic germ; ES, embryonic stem; ERas, ES cell-expressed Ras; ECAT, ES cell-associated transcript; GSK, glycogen synthase kinase; LIF, leukaemia inhibitory factor; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PDK1, phosphoinositide-dependent kinase 1; PGC, primordial germ cell; PH domain, pleckstrin homology domain; PI3K, phosphoinositide 3-kinase; PIKK, phosphoinositide kinase-related kinase; PMEF, primary mouse embryonic fibroblast; PTEN, phosphatase and tensin homologue deleted on chromosome 10; RalGDS, Ral guanine nucleotide dissociation stimulator; Rheb, Ras homologue enriched in brain; TSC, tuberous sclerosis complex
- © 2005 The Biochemical Society