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PTEN function in mammalian cell size regulation Stéphanie A Backman*, Vuk Stambolic† and Tak W Mak‡ The PTEN tumor suppressor gene is a lipid phosphatase that negatively regulates cell survival mediated by the phosphatidyl inositol 3′ kinase-protein kinase B/Akt signaling pathway. Recent in vivo studies have revealed a novel role for PTEN in the size control of neurons. Dysregulation of cell growth control by PTEN is associated with the neurological disorder Lhermitte-Duclos disease. PTEN may regulate cell size through effects on protein translation. Addresses Department of Medical Biophysics, University of Toronto and Ontario Cancer Institute, 610 University Avenue, Toronto, M5G 2M9, Canada *e-mail: [email protected] † e-mail: [email protected] ‡ e-mail: [email protected] Current Opinion in Neurobiology 2002, 12:516–522 0959-4388/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 14 August 2002 Abbreviations 4E-BP eIF4E-binding protein ATP adenosine triphosphate CNS central nervous system eIF eukaryotic translation initiation factor LDD Lhermitte-Duclos disease MMAC/TEP1 mutated in multiple advanced cancers/transforming growth factor β enhanced protein 1 NGF nerve growth factor PDK1 phosphoinositide-dependent kinase 1 PI3K phosphatidylinositol 3′ kinase phosphatidylinositol (3,4,5) triphosphate PIP3 PTEN phosphatase and tensin homolog, deleted on chromosome 10 p70S6K p70 S6 kinase TOR target of rapamycin TSC tuberous sclerosis complex

Introduction PTEN (named for its homology to phosphatases and tensin and the deletion of its gene on chromosome 10 in human cancers) has been under intense study for the last five years because of its role as a tumor suppressor in various advanced cancers [1–3]. Genetic and biochemical studies have revealed an important role for this gene in programmed cell death and proliferation [4–6]. A novel function for PTEN in the control of cell size has recently been discovered and is associated with severe pathological consequences in the central nervous system (CNS). In this review, we describe cell growth control by PTEN in the mammalian brain. We further discuss PTEN’s ability to regulate known cell size regulators involved in protein translation, including phosphatidylinositol 3′ kinase (PI3K) effectors, as one of the possible mechanisms of neural cell size determination.

PTEN: a suppressor of PI3K-PKB/Akt-mediated cell survival PTEN was initially identified as a tumor suppressor mutated in glioblastomas, breast, prostate and kidney cancers [1,2].

This gene was identified by three independent groups and is also referred to as MMAC/TEP1 (mutated in multiple advanced cancers/transforming growth factor β enhanced protein) [2,3]. Germline mutations of PTEN cause Cowden’s syndrome, a disease characterized by hamartomas (benign growths) of multiple tissue types, especially skin and brain, and a predisposition to malignancies of the brain, breast and thyroid. Lhermitte-Duclos disease (LDD) is a rare CNS manifestation of Cowden’s disease caused by a hamartomatous overgrowth of the cerebellum [7]. Germline mutations in PTEN’s phosphatase domain have been identified in LDD patients [8–11]. Clinical features of LDD include macrocephaly, ataxia, seizures, hydrocephalus, raised intracranial pressure, progressive headaches, cranial nerve deficits and mental retardation [7,11,12]. The PTEN protein contains a phosphatase domain, a region with high homology to tensin and a protein interaction PDZ (PSD95/Discs Large/ZO-1) binding site at its carboxy (C)-terminus [1,2]. Crystal structure analyses of PTEN revealed that it also possesses a C2 lipid-binding domain [13]. Although germline and sporadic mutations that inactivate PTEN are scattered across the entire gene, point mutations frequently target the phosphatase domain [14]. The elucidation of PTEN’s ability to antagonize cell survival mediated by the PI3K-protein kinase B/Akt (PI3K-PKB/Akt) signaling pathway was an important step towards understanding PTEN function [4,15]. Certain components of this pathway are amplified or overexpressed in various human and mouse tumors [16,17]. The PI3K/PTEN intracellular signaling cascade has been reviewed elsewhere [17–19]. Briefly, growth factor stimulation of cells causes activation of PI3K and an increase in cellular levels of the membrane phospholipid phosphatidylinositol (3,4,5) triphosphate (PIP3), a key mediator of cell survival (Figure 1). Elevated PIP3 levels result in membrane recruitment, phosphorylation and activation of PKB/Akt, a potent survival effector. Despite homology to protein phosphatases, PTEN dephosphorylates the D3 position of the inositol ring of PIP3 and negatively regulates PKB/Akt activity. PTEN’s effects on cellular processes likely extend beyond its effect on cell survival, considering PI3K-PKB/Akt’s involvement in the regulation of other cellular processes, such as protein translation and glucose metabolism [17,20].

A novel role for PTEN identified in Drosophila Studies in Drosophila melanogaster reveal a novel role for PTEN in the control of tissue growth [21–23]. The phenotypes of flies carrying mutations for various components of the PI3K-PKB/Akt pathway have shown that this pathway positively controls cell number and cell size [24–27]. Consistent with its role as an antagonist of this pathway, Drosophila PTEN (dPTEN) loss-of-function mutants

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Figure 1 PTEN negatively regulates PKB/Akt-mediated cell survival. Binding of growth factors (GFs), for example platelet-derived growth factor, insulin or NGF, to receptor tyrosine kinases (RTKs) activates PI3K. Activated PI3K phosphorylates (P) phosphatidylinositol (4,5) biphosphate (PIP2) to produce PIP3, which recruits PKB/Akt to the plasma membrane where it is phosphorylated and activated by PDK1. PTEN prevents PKB/Akt-mediated cell survival by dephosphorylating PIP3. PKB/Akt also regulates protein translation and glucose metabolism. Target substrates of PKB/Akt are listed in brackets. Bad, Bcl-XL/Bcl2 associated death factor; FKHR/AFX, forkhead family transcription factors; Glut4, glucose transporter 4; GSK3, glycogen synthase kinase 3; IKK, IκBα-kinase kinase.

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display increased cell and organ size; overexpression of dPTEN yields the opposite phenotype. Additionally, PTEN negatively regulates cell survival and proliferation in Drosophila. Drosophila PKB/Akt (dPKB/Akt) mutations fully suppress the cell size phenotype caused by loss of dPTEN [23]. Further, although mutations of the insulin receptor result in decreased cell size, this mutation does not suppress the dPTEN phenotype, strongly suggesting that dPTEN acts downstream of growth factor signaling [23].

PTEN deletion in the brain: a role for PTEN in mammalian cell growth control PTEN-null fibroblasts, thymocytes and embryonic stem cells do not display any apparent defects in cell size (V Stambolic, A Suzuki, T Mak, unpublished data). Given that the cell growth phenotypes of some Drosophila mutations are not always paralleled by their mammalian counterparts [28], uncertainty surrounded the issue of whether PTEN regulated the volume of any mammalian cells. However, recent in vivo studies by our group [29••] as well as by Kwon et al. [30••] exploring PTEN function in the brain demonstrate a physiological role for PTEN in mammalian cell size regulation. The Cre-loxP system [31] was used to inactivate PTEN in the mouse brain, resulting in deletion of PTEN in granule neurons of the cerebellum and the dentate gyrus. Loss of PTEN in these cells resulted in seizures, ataxia and premature death. Brains from these mice were enlarged, particularly the cerebellum. Interestingly, no tumors were observed and levels of apoptosis and proliferation appeared normal. However, the somas of PTEN-null neurons were up to two times larger than their control counterparts. Thus, loss of PTEN caused neuronal cell

bodies to grow beyond their normal size in vivo, contributing to brain enlargement. Consistent with the phenotype of dPTEN chimeric fly eyes and wings [21–23], regions exhibiting mosaic loss of PTEN revealed that regulation of neuron size by this gene is cell-autonomous. Significantly, the neurological symptoms and macrocephaly of the brain-specific PTEN mutant mice resemble those of patients suffering from LDD. Similarly to PTEN mutant mice, the primary source of symptoms in LDD patients is a cerebellar growth. This lesion is characterized by the presence of hypertrophied granule cells, a low proliferative index and elevated PKB/Akt phosphorylation [7,32], all features of the PTEN mutant mice. Further support for the ability of PTEN to regulate neuronal growth comes from an independent study in which PTEN was deleted in CNS stem/progenitor cells [33••]. PTEN loss in neural stem cells also resulted in brain enlargement, due to an overall increase in the size of neural cells, accompanied by increased proliferation and decreased apoptosis. Using neurosphere cultures, Groszer et al. [33••] demonstrated that PTEN-null neuronal progenitors displayed increased proliferation and size. Furthermore, these cells had a shortened cell cycle and underwent more self-renewing divisions than their wild-type counterparts.

Does PTEN function depend on differentiation? Results from brain-specific PTEN mutants suggest that PTEN function varies depending on the differentiation status of cells. Enhanced proliferation could be detected in PTEN-null neural stem cells but not in fully differentiated granule neurons. Nevertheless, differentiated neurons lacking PTEN display abnormal cell growth. Taken together, these observations imply that PTEN’s effects on proliferation

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Figure 2

reaching the minimal threshold size. In the proposed model described above, PTEN-null differentiated neurons grow beyond their normal size, but this enhanced growth cannot translate into increased proliferation (Figure 2). The effects of PI3K/PTEN signaling in other differentiated tissues support such a view. Overexpression of constitutively active PI3K in terminally differentiated myocytes causes heart enlargement via an increase in cell size without affecting the cell number or proliferation [39••]. Furthermore, fully differentiated PTEN-null bristle cells in the fly wing are enlarged with no evidence of increased proliferation [23].

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Hypothetical model of PTEN’s differentiation-dependent roles. The ability of PTEN to regulate cell growth influences cell cycle entry of undifferentiated cells. In this model, PTEN continues to regulate growth of post-mitotic, undifferentiated cells, but because these cells are no longer equipped to initiate cell cycle entry, PTEN’s effects on cell growth cannot influence proliferation. T bar represents inhibition; arrows denote activation.

are restricted to mitotic, undifferentiated cells, whereas in post-mitotic, differentiated cells, PTEN has the ability to influence cell growth. This proposed model is strongly supported by the fact that enhanced proliferation detected upon PTEN deletion is restricted to mitotic regions; no ectopic proliferation could be found in differentiated cells that have withdrawn from the cell cycle [29••,30••,33••]. Similarly, dPTEN overexpression in Drosophila negatively regulates proliferation of mitotic eye cells, but not cells that have begun differentiation [21]. Furthermore, overexpression of a constitutively activated mutant of PKB/Akt in mouse brains contributed to the development of brain tumors (which are typically highly proliferative) only when introduced into neural stem cells, not when introduced into differentiated neural cells [34••]. In light of the fact that cell size is an integral part of the cell cycle, PTEN may influence proliferation through its effects on cell growth. Cells must grow to a minimal threshold size before progressing through the cell cycle [35,36]. Hypothetically, the increased growth rate associated with loss of PTEN allows cells to reach the minimal threshold size more rapidly, accelerating cell cycle progression and thus increasing the overall cell number. Consistent with such a notion, accelerated G1/S progression in PTEN-null embryonic stem cells has been described [6]. So, why are these proliferative defects not detected in differentiated neurons lacking PTEN? Possibly because elements critical for cell cycle progression are downregulated during terminal differentiation [37,38]. Thus, unlike neural stem cells, differentiated cells are not equipped with the necessary machinery to initiate cell cycle entry upon

An additional mechanism by which PTEN may regulate the cell cycle machinery is through regulation of the cyclin-dependent kinase inhibitor p27KIP1. Cell cycle profile defects observed in PTEN-null neural and embryonic stem cell lines are paralleled by dysregulation of this cell cycle regulator [6,40]. Any influence that PTEN may have on p27KIP1 is likely cell-type-specific, because the expression pattern of p27KIP appears normal in cerebellar granule neurons and primary mouse embryonic fibroblasts lacking PTEN [6,30••] (V Stambolic, unpublished data). The progressive nature of PTEN function in neuronal differentiation is underscored by the fact that neuron growth defects observed in the brain-specific PTEN mutant mice become much more pronounced as neuronal differentiation proceeds [29••,30••]. Interestingly, even though cell fate determination is unaffected by the absence of PTEN [29••,30••,33••] (S Backman, T Mak, unpublished data), expression of this gene is induced during neuronal differentiation [41]. Thus, PTEN’s role in postmitotic neuronal growth may be independent of other aspects of differentiation.

PTEN, cell growth and human hamartoma diseases Increased proliferation of PTEN-deficient progenitor stem cells and enhanced growth of their differentiated progeny closely resembles hamartomas associated with Cowden’s disease. Hamartomas, such as LDD, constitute benign overgrowths of mature cells indigenous to the tissue of origin [42]. Although Cowden’s disease manifests itself in both fully differentiated and actively proliferating tissues (such as skin, breast and thyroid), cell hypertrophy can only be observed when the affected tissue is post-mitotic (e.g. cerebellar granule neurons in LDD). Furthermore, an increased number of mature neurons is frequently detected in LDD, but mitotic indices are low [32], implying increased proliferation of progenitor cells (as suggested in [30••]). However, given that activation of the PI3K pathway protects neurons from apoptosis, a decrease in the extensive amount of apoptosis that occurs in the developing brain [43] could also contribute to the expansion of progenitors and the overgrowth of neurons observed in LDD. Another hamartoma condition caused by mutations in cell size regulatory genes is tuberous sclerosis complex (TSC),

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Figure 3 The full activation of p70S6K likely requires input from both PI3K and TOR signalling. S6K, which can be activated by PDK1 and TOR, influences protein translation by promoting the translation of 5′-terminal oligopyrimidine tract-containing mRNAs (5′TOP mRNAs — the majority of which encode components of the protein translation machinery) perhaps through S6 phosphorylation. S6K may also activate eIF4B. TOR is sensitive to amino acid (AA) and ATP levels and may be phosphorylated and activated by PKB/Akt. Phosphorylation of 4E-BP by TOR frees eIF4E to join the translation initiation complex. eIF4GI is also activated by PI3K signaling.

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caused by mutations in Tsc1 and Tsc2 [42,44,45••,46••]. Affected tissues in TSC and LDD overlap but are not identical. However, a notable similarity is the presence of giant differentiated cells in both conditions. Interestingly, Drosophila loss-of-function and overexpression studies provide strong evidence for a genetic interaction between the Tsc genes and the PI3K/PTEN pathway [45••,46••]. On the basis of these findings, it is tempting to speculate that hypertrophy caused by dysregulation of the PI3K pathway is associated with some manifestations of TSC. Further investigations of the functional and genetic interactions between the PI3K/PTEN pathway and the Tsc genes in mammalian cell size control are required to explore this possibility. Hypertrophy associated with loss of PTEN may extend to other neural cell types and CNS disorders. Limited evidence from cell culture studies suggests that the PI3Kactivating ligands nerve growth factor (NGF) and insulinlike growth factor 1 promote hypertrophy in sympathetic ganglion cell neurons and Schwann glial cells, respectively [47,48]. PTEN mutations have recently been identified in Proteus and Proteus-like syndrome, conditions associated with benign growths and neuronal hypertrophy [49–52]. Furthermore, enlarged astrocytes are often present in glioblastomas, tumors in which PTEN is frequently mutated [1,2,53,54]. Whether hypertrophy is directly caused by, or is a reactive consequence of lesions ensuing from the loss of PTEN is unknown.

Does PTEN control cell growth through regulation of protein translation? One possible explanation for the function of PI3K/PTEN signaling in regulation of cell growth is its ability to regulate protein translation. Several molecules involved in the control of protein synthesis, including p70 S6 kinase (p70S6K), target of rapamycin (TOR) and eukaryotic translation initiation factor (eIF)4E-binding protein (4E-BP) are downstream targets of PI3K signaling [16] (Figure 3) and known regulators of cell size [55,56•,57•,58•]. PI3K/PTEN signaling may influence protein translation through PKB/Akt-mediated phosphorylation of TOR [16]. TOR is a key regulator of protein translation and is sensitive to nutrient and ATP availability [59••]. Through its kinase activity, TOR activates p70S6K, a regulator of the 40S ribosomal protein S6, and inactivates 4E-BP, an inhibitor of eIF4E. In addition, PI3K/PTEN signaling could regulate p70S6K through phosphoinositide-dependent kinase 1 (PDK1), a PIP3-dependent kinase that can directly phosphorylate p70S6K [60,61]. It is also possible that the PI3K/PTEN pathway controls cell growth through regulation of other growth factor-induced initiation factors including eIF4A, eIF4B and eIF4GI [16]. p70S6K is believed to preferentially mediate translation of mRNAs encoding ribosomal proteins and to affect the overall rate of protein synthesis [16]. Deletion of p70S6K in mice and flies results in smaller overall body size and reduced organ size, including the brain [55,62,63••]. Although other

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components of the PI3K and TOR pathways regulate organ size through changes in both cell number and cell size, the small organs observed in p70S6K-deficient flies result solely from a decrease in cell size [55], implicating p70S6K as a specific cell size regulator downstream of these pathways. It appears that full activation of p70S6K requires input from TOR signaling as well as PI3K/PTEN signaling (potentially through PDK1) [16]. Strong genetic evidence links the PI3K/PTEN pathway to both TOR and PDK1. Enlargement of dPTEN mutant cells in the wing requires dTOR function [56•], whereas the viability of dPTEN mutants can be rescued by mutations of dPDK1 [64•]. Furthermore, genetic epistasis indicates that dS6K is downstream of both PDK1-mediated and TOR-mediated growth regulatory events [56•,64•]. On the basis of recent studies of PKB/Akt function in Drosophila demonstrating its essential role in interpreting PI3K signals, it is likely that the cell growth defects caused by PTEN loss are mediated via PKB/Akt [65••]. Consistent with such a notion, enlarged PTEN-null neurons display hyperphosphorylation of PKB/Akt [29••,30••] and PKBα/Akt1-deficient mice are smaller than their littermate controls [66••,67••], implicating PKB/Akt in mammalian growth. Detailed investigation of cell size defects in PKBα/AKT1-null and TOR-deficient mice will help to determine the relevance of these molecules in mammalian cell growth.

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Conclusions and future directions Recent studies have demonstrated a novel role for the tumor suppressor PTEN in the control of neuron size. Regulation of cell growth by PTEN is cell-autonomous and depends on the cell type and its differentiation status. Significantly, dysregulation of cell growth through loss of PTEN is associated with development of LDD, a human neurological disorder caused by germline PTEN mutations. Molecular mechanisms for PTEN’s regulation of cell size potentially involve control of protein translation and the Tsc gene products, revealing an intriguing cellular signaling network implicated in both cell growth and tumorigenesis. Further investigation of PTEN function in cell growth control as well as a better understanding of PTEN’s role in cell differentiation will provide critical insights into its physiological role in regulation of cellular homeostasis and ultimately tumor suppression.

Acknowledgements This work is supported by the National Cancer Institute of Canada, the Canadian Breast Cancer Research Initiative and the Terry Fox Foundation. SA Backman holds an Ontario Graduate Scholarship. We acknowledge Patrick Shannon and Abhijit Guha for thoughtful discussions and Megan Cully for critical review of this manuscript.

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