|1 November 1999, Volume 18, Number 45, Pages 6094-6103|
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|Modulation of cellular apoptotic potential: contributions to oncogenesis|
|Vuk Stambolic1,2, Tak W Mak1,2 and James R Woodgett2|
1Amgen Institute, 620 University Avenue, Toronto, Ontario, Canada M5G 2C1
2Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9
Correspondence to: James R Woodgett, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9
The importance of apoptosis as a natural means to eliminate unwanted or damaged cells has been realized over the past decade. Many components required to exercise programmed cell death have been identified and shown to pre-exist in most, if not all, cells. Such ubiquity requires that apoptosis be tightly controlled and suggests the propensity of cells to trigger the cellular death machinery can be regulated. Recently, several signaling pathways have been demonstrated to impact the apoptotic potential of cells, most notably the phosphatidylinositol 3' kinase (PI3'K) pathway. The 3' phosphorylated lipid products generated by this enzyme promote activation of a protein-serine kinase, PKB/AKT, which is necessary and sufficient to confer cell PI3'K-dependent survival signals. The relevance of this pathway to human cancer was revealed by the recent finding that the product of the PTEN tumor suppressor gene acts to antagonize PI3'K. This review focuses on the regulation and mechanisms by which PKB activation protects cells and the oncologic consequences of dysregulation of the pathway.
apoptosis; cell survival; PI3'K; PTEN
Despite its late recognition as a fundamental process in multicellular organisms, apoptosis has rapidly become integrated into the understanding of a wide variety of biological events and has instituted appreciation of the delicate balances within cells that dictate ongoing viability or termination (Steller, 1995). It had long been recognized that tumors are associated with considerable cell death as well as proliferation. However, the realization that the primary means by which gamma irradiation and most chemotherapeutics killed cells was by inducing a suicide response revolutionized thinking about how such therapies might be improved and how cells might evade their action. A similar watershed in thinking was instigated by the finding that the bcl2 oncogene acted not by promoting growth, as was seemingly the case for previously discovered oncogenes, but by suppressing cell death (Korsmeyer, 1999). These discoveries pointed to an equally important role for control of cell death as for control of cell division and brought about a surge in efforts to identify genes involved in the process of programmed cell death. These efforts were accelerated by the genetic characterization of apoptosis in the nematode, largely through the work of Hengartner and Horvitz (1994) and also Metzstein et al. (1998), which provided key insights into the nature of the positively and negatively acting gene products.
With emphasis placed on the identities of the `executioners' and the `appeal judges', there was rapid progress in characterizing the caspase family of proteases activated during apoptosis and additional relatives and antagonists of Bcl2 such as Bcl-Xl, Bax, Bag and Bad (reviewed in Li and Yuan, 1999; Reed, 1998). Simultaneously, work in a variety of biological systems began to uncover differential sensitivities of cells to survival in culture depending on the presence of particular factors in the media (Dudek et al., 1997). These experiments indicated that extracellular molecules could influence cellular viability and provided models to facilitate dissection of the pathways induced by such factors.
The viability of cultures of primary neurons has long been known to be highly dependent upon particular factors, such as neurotrophins (such as NGF, CNTF and BDNF). Likewise, several hematopoietic cell lines were known to be dependent upon certain growth factors such as IL-3 and GM-CSF. Ectopic expression of certain genes was found to prolong neuronal survival (such as dominant negative c-Jun; Ham et al., 1995) but most effort was focused on the mechanisms by which these growth factors enhanced survival. Yao and Cooper (1996) noted that inhibition of phosphatidylinositol 3' kinase (PI3'K) induced apoptosis. At the time, the effectors of this lipid kinase were thought to be largely related to metabolic controls such as protein synthesis, vesicular transport and sugar transport (Vanhaesebroeck et al., 1997). Furthermore, the only inhibitors of PI3'K (wortmannin and LY294002) were known to effect other cellular processes. However, there was a strong correlation between activation of PI3'K and protection from cell death.
This association was strengthened by a different avenue of work investigating the effects of the c-Myc oncoprotein on apoptosis. Normally, overexpression of c-Myc is associated with cell proliferation. In quiescent cells, c-Myc levels are undetectable but are rapidly elevated upon mitogenic stimulation. Serum-deprivation results in a decrease of Myc expression and is accompanied by ordered exit of cells from the cell cycle. However, forced expression of Myc induced apoptosis rather than quiescence of cells. Thus, serum deprivation of cells expressing a constant level of Myc resulted not in exit from the cycle but apoptosis (Evan et al., 1992). By screening for constituents in serum capable of blocking this death, Evan's group identified factors such as IGF-1 and PDGF as being protective (Harrington et al., 1994). These factors all induced activation of PI3'K.
Progress in determining the molecular mechanism by which PI3'K was able to transduce a survival signal was significantly enhanced by the discovery that a previously identified protein-serine kinase termed protein kinase B (PKB or AKT) was activated by the 3' phosphorylated products of PI3'K (Burgering and Coffer, 1995; Franke et al., 1995; Kohn et al., 1995). An oncogenic variant of PKB had been identified in 1991 as a retrovirally transduced fusion of PKB with gag sequences resulting in a constitutively activated enzyme (Bellacosa et al., 1991). Expression of gag-PKB/v-Akt in cells was shown to confer a similar degree of protection as the agonists of PI3'K. For example, introduction of gag-PKB into cerebellar neurons (Dudek et al., 1997) or fibroblasts (Kauffmann-Zeh et al., 1997) increased viability in the absence of serum. Furthermore, expression of catalytically inactive PKB reduced the viability of the cells in the presence of survival factors (Dudek et al., 1997; Stambolic et al., 1998). In other cell types, expression of activated PKB protected cells against UV irradiation (Kulik et al., 1997) and IL-3 withdrawal-induced death (Songyang et al., 1997).
Regulation of PKB
All PKB gene products encode a polypeptide of approximately 55 - 60 kDa containing an N-terminal pleckstrin homology (PH) domain and a serine/threonine kinase catalytic domain (reviewed in Coffer et al., 1998). The PH domain exhibits affinity for 3' phosphorylated lipids although the exact preferences are unclear (in part due to the importance of the context of presentation of the lipids to the protein) (James et al., 1996; Klippel et al., 1997; Takeuchi et al., 1997>). Activation of PKB requires an intact PH domain but this is insufficient for function. For catalytic activation the enzyme must undergo a conformational shift that occurs upon phosphorylation of two residues, a threonine in the catalytic loop (T308 in PKB) and a serine close to the carboxyl terminus (S473 in PKB) (Alessi et al., 1996). These residues are conserved in all PKBs (including Drosophila and C. elegans) except for a splice variant of the PKB gene (Konishi et al., 1995) which encodes a truncated polypeptide that lacks the C-terminal phosphorylation site (a longer splice variant encoded by the same gene does contain a serine homologous to S473) (Brodbeck et al., 1999; Nakatani et al., 1999).
Phosphorylation of the two activatory sites is required for full activation of PKB (Alessi et al., 1996). Mutation of either to a non-phosphorylatable residue significantly reduces activity and mutation of both eliminates enzymatic activity. Phosphorylation of the two residues is dependent upon PI3'K activity and is inhibited by wortmannin. Two enzyme activities that independently phosphorylate T308 and S473 of PKB, termed polyphosphatidylinositide-dependent protein kinases 1 and 2 (PDK1 and PDK2), have been characterized. Substitution of the activation site residues with aspartic acid (with the aim of mimicking negatively charged phosphates) results in a partially activated enzyme that is neither further activatable by agonists of PI3'K or inhibitable by wortmannin or LY294002. Interestingly, following phosphorylation by the PDKs, a significant fraction of active PKB molecules translocate to the nucleus (Andjelkovic et al., 1997; Meier et al., 1997). The sequence of events required for induction of PKB is thus complex (see Figure 1). Following activation of PI3'K (for example, by its own translocation to the plasma membrane via recruitment of its regulatory subunit's SH2 domains to phosphotyrosine docking sites on activated receptors or receptor adaptors), a complex assembles, drawn to newly generated 3' phospholipid microdomains. The complex consists of PKB and its two processing kinases, the PDKs. Following phosphorylation the activated PKB is drawn into the cellular interior and nucleus. Since aspartate mutants of the activation sites are PI3'K independent and short circuit this process, membrane binding of PKB acts simply to allow its co-localization with the processing enzymes, the PDKs. Phosphorylation by these enzymes is thought to induce a conformational change that both reduces affinity for 3' phosphorylated lipids and opens up the catalytic cleft, allowing access to substrates.
PDKs have other roles
PDK1 was purified by affinity chromatography, relying on its affinity for PKB (Alessi et al., 1997b; Stokoe et al., 1997). The enzyme consists of a C-terminal PH domain and a protein kinase catalytic domain (and little else) (Alessi et al., 1997a). PDK1 appears to be constitutively active in cells. When purified, the protein does not require phospholipids or any other co-factors for expression of activity (Alessi et al., 1997b). Indeed, PDK1 phosphorylates other protein kinases in addition to PKB, including various PKCs (Dong et al., 1999; Le Good et al., 1998), p70 S6 kinase (Alessi et al., 1998; Pullen et al., 1998) and cAMP-dependent protein kinase (PKA) (Cheng et al., 1998). In all cases, the targeted phosphorylation site is deep within the kinase domain and, in the case of PKA, plays a structural role (Cheng et al., 1998). The PDK1 site on PKA appears to be phosphorylated upon synthesis and initial folding of the enzyme. These data support a tonic function for PDK1 in the processing of certain other protein kinases and may partly explain the unusual phenotypes associated with mutation of the PDK1 homologue in Drosophila (DSTPK61) (Belham et al., 1999). The question remains, why is PDK1 phosphorylation of PKB dependent upon 3' phosphorylated lipids. The most likely explanation is that binding of 3' phospholipids by the PH domain of PKB reveals the PDK1 site. Thus, phosphorylation can only occur at the membrane.
Elucidation of PDK2 has been more problematic. Two distinct possibilities have been proposed. Alessi et al. performed a two hybrid screen with PDK1 and isolated a C-terminal 77 amino acid fragment of PRK2, yet another protein-serine kinase. They found that co-expression of PDK1 with the PRK2 fragment modified it's substrate specificity, enabling it to phosphorylate the PDK2 site on PKB (Balendran et al., 1999). Although the PRK2 fragment does not exist physiologically, the group hypothesized that another molecule may act similarly and convert PDK1 into a PDK2 like activity. This novel idea has yet to be shown to have physiological significance but, if true, would remove the necessity for an independent PDK2 enzyme. A prediction of this model would be co-regulation of both sites of phosphorylation on PKB (T308 and S473). Phosphorylation of each is dependent upon PI3'K. However, mutation of one site does not affect phosphorylation of the other and certain kinase inhibitors block T308 phosphorylation without affect on S473 (B Hemmings, personal communication).
A distinct PDK2 candidate emerged from study of a protein kinase isolated by virtue of its affinity for the intracellular domain of -integrins. Integrin-linked kinase (ILK) has an unusual catalytic domain located C-terminally to ankyrin repeats (Hannigan et al., 1996). Between these features lies a sequence with homology to phospholipid binding domains. ILK activity was found to be induced upon integrin ligation and to be PI3'K-dependent (Delcommenne et al., 1998). Furthermore, expression of ILK in cells resulted in activation of PKB and its phosphorylation on S473. In vitro ILK can incorporate phosphate into PKB, specifically at this residue. Similar data have been obtained using Drosophila ILK which phosphorylates Drosophila PKB at serine 505 (analogous to S473 in the mammalian protein) (A Ali and J Woodgett, unpublished observations) These data raise the possibility of a mechanism to couple PKB activation with matrix attachment. When epithelial cells lose contact with other cells, they undergo a form of apoptosis termed `anoikis' (Frisch and Francis, 1994). Expression of activated mutants of PKB suppresses this death (Fujio and Walsh, 1999; Khwaja et al., 1997>). Given the key role of integrins in matrix signaling, it is tempting to speculate that ILK provides the integrin-mediated survival signal when cells are anchorage-dependent.
Targets of PKB
Knowing that expression of an activated allele of PKB is sufficient to confer a high degree of protection from apoptosis, there has been intensive effort to identify the important substrates. One of the first targets identified was glycogen synthase kinase-3 (GSK-3), which is inactivated by PI3'K signaling and can be directly phosphorylated by PKB in vitro (Cross et al., 1995). Overexpression of GSK-3 can induce cell death (Pap and Cooper, 1998). However, lithium inhibits this enzyme without apparent effects on apoptotic propensity (Stambolic et al., 1996).
Several other gene products have been proposed that play a direct role in promoting apoptosis. In particular, the pro-apoptotic, Bcl-2 related protein, Bad, contains a serine residue within a consensus sequence recognized by PKB. Bad dimerizes with Bcl-Xl and smothers its anti-apoptotic capacity. Co-expression of Bad and PKB leads to the former protein becoming phosphorylated (at serine 136) which facilitates binding to 14-3-3 proteins (Datta et al., 1997; del Peso et al., 1997; Gajewski and Thompson, 1996; Yaffe et al., 1997; Zha et al., 1996). As a consequence of phosphorylation, Bad dissociates from Bcl-Xl allowing that protein to exert protective effects. In essence, phosphorylation of Bad by PKB neutrilizes the suppressive effects of Bad on Bcl-Xl.
The physiological significance of this mechanism is somewhat limited by the narrow expression profile of Bad, which is largely confined to hematopoietic cells. Due to the relatively low endogenous levels of Bad, it has also been difficult to demonstrate the effect without resorting to transient overexpression. Further, activation of PKB by cytokines and induction of Bad phosphorylation can be uncoupled (Craddock et al., 1999; Hinton and Welham, 1999; Scheid and Duronio, 1998).
The executioners of apoptosis include a family of proteases that cleave after aspartate residues, the caspases (Nunez et al., 1998). These largely fall into one of two classes: upstream caspases that are coupled to regulatory machinery (such as FLICE which associates with the Fas receptor adaptor, FADD) and those that are activated by proteolytic cleavages catalysed by the activated upstream caspases (effector caspases). Caspase 9 is an upstream caspase that is activated upon stiimulation of cells by agents such as TNF and other cellular stresses. Human caspase 9 contains a PKB consensus sequence and can be phosphorylated by PKB in vitro, resulting in inactivation of its protease activity (Cardone et al., 1998). Further, phosphorylation of caspase 9 at the PKB site can be demonstrated in human cells. By phosphorylating caspase 9, PKB would therefore reduce the capacity of a cell to induce the proteolytic activation of certain downstream caspases. The significance of this mechanism is unclear, however, since a major fraction of caspase 9 molecules would have to be phosphorylated to impact the caspase autolytic cascade. Further, the site of phosphorylation in human caspase 9, which has been mapped to Ser 196, lies within a V8 proteolytic fragment KLRRRFSSLHFMVE (Ser 196 underlined) (Cardone et al., 1998). However, the analogous peptide from murine caspase 9, KLEHRFRWLRFMVE (R Hakem, personal communication) does not contain a phosphorylatable residue in the position corresponding to Ser 196.
Perhaps the most compelling anti-apoptotic targets identified to date is the family of transcription factors than include the Forkhead-like (FKHR)/Afx proteins (Biggs et al., 1999; Brunet et al., 1999; Guo et al., 1999; Kops et al., 1999; Nakae et al., 1999; Rena et al., 1999). Among the genes induced by these factors are various pro-death molecules including fas ligand. For example, PKB-dependent phosphorylation of FKHRL1 at Thr 32 and Ser 253 promotes its association with 14-3-3 proteins. This is associated with nuclear export denying access of this transcription factor to its DNA targets (Brunet et al., 1999). Thus, activation of PKB effectively shuts down a genetic program that includes genes that can trigger cellular suicide. The first clue that this family might be regulated by PKB came from studies in the nematode (see Figure 1). Genetic analysis of the daf2/insulin/IGF1 receptor revealed a suppressor termed daf16, which, when cloned, was found to encode a forkhead-related transcription factor (Lin et al., 1997; Ogg et al., 1997). Together, the biochemical and genetic data suggest that several members of the forkhead family are important targets for PI3'K mediated survival signaling. Even so, there are likely many additional PKB substrates that mediate its survival promoting effects that remain to be identified.
Importance of PI3'K signaling in cancer
The role of phosphatidylinositol metabolism in tumorigenesis was first implied a number of years ago by the findings that products of viral oncogenes pp60 v-src and polyoma virus middle T antigen associate with an intracellular phosphatidylinositol kinase activity (Sugimoto et al., 1984; Whitman et al., 1985). It was shown that the regulatory subunit of phosphatidyinositol 3' kinase (PI3'K) was able to directly interact with these oncogenes and was responsible for the associated PI3' kinase activity (Carpenter et al., 1990; Kaplan et al., 1987; Serunian et al., 1990). In support of the role of PI3'K in cellular transformation, an oncogenic form of the catalytic subunit of PI3'K was cloned from a retrovirus that causes hemangiosarcomas in chickens (Chang et al., 1997) and shown to induce transformation of chicken embryo fibroblasts. More recently, amplification of the human gene encoding the p110 catalytic subunit of PI3'K in ovarian cancer tissue samples and cell lines has been described (Shayesteh et al., 1999), as well as the ability of the activated form of PI3'K to cause transformation of 3T3 cells (Jimenez et al., 1998). PI'3K functions in multiple cellular signaling pathways and is implicated in regulation of cell proliferation, survival and adhesion, organization of the cytoskeleton and glucose metabolism (reviewed in Rameh and Cantley, 1999; Leevers et al., 1999; Fruman et al., 1998; Shepherd et al., 1998). A role for PI3'K in tumorigenesis is underscored by the identification of activating mutations in both upstream and downstream components of PI3'K signaling pathways in human cancer. For example, amplification of members of the receptor tyrosine kinase family capable of activating PI3'K such as platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) genes have been demonstrated in glioblastoma (Chaffanet et al., 1992; Smits and Funa, 1998). Identification of PKB/Akt, a transforming oncogene that causes thymic lymphomas in mice (see above), as a major target of PI3'K signaling further supports the importance of PI3'K/PKB in cancer. Moreover, in humans, overexpression of PKB has been demonstrated in a proportion of ovarian (Cheng et al., 1992), pancreatic (Cheng et al., 1996) and breast cancers (Bellacosa et al., 1995). ILK has also been found to be overexpressed in ovarian and breast cancer and is able to transform epithelial cells (S Dedhar, personal communication; Wu et al., 1998). Thus, changes in the activity of PI3'K signaling pathway(s) due to amplification and/or upregulation of its components could result in complex cellular outcomes resulting in cellular transformation and development of cancer (Table 1).
The recent identification of the molecular mechanism of action of the tumor suppressor gene PTEN/MMAC1/TEP1 (PTEN herein; see below) has offered new insights into the involvement of PI3'K-regulated signaling pathways in a large fraction of human tumors. PTEN was originally identified as a candidate tumor suppressor gene frequently deleted at chromosome 10q23 in a number of advanced tumors such as glioblastoma, prostate, kidney and breast carcinoma (Li et al., 1997; Steck et al., 1997). PTEN was also independently discovered in a search for novel tyrosine phosphatases and named TEP1 (Li and Sun, 1997). A systematic search for the involvement of PTEN alterations in human cancer by a number of groups demonstrated a significant rate of PTEN mutations in high-grade but not low-grade glioblastomas (Bostrom et al., 1998; Liu et al., 1997; Rasheed et al., 1997; Wang et al., 1997), prostate (Cairns et al., 1997), and thyroid (Dahia et al., 1997) tumors, as well as in breast (Li et al., 1997; Steck et al., 1997; Teng et al., 1997) and melanoma (Guldberg et al., 1997) cell lines. In contrast to other tumors where PTEN mutations are frequent in the advanced phases of the disease, PTEN mutations also occur at all stages of endometrial cancer (Risinger et al., 1997, 1998; Tashiro et al., 1997), suggesting a potential involvement of PTEN in the process of tumor initiation in this organ.
In addition to frequent mutations in sporadic tumors, germline mutations of PTEN are believed to cause three related autosomal-dominant hamartoma syndromes: Cowden syndrome (Liaw et al., 1997), Bannayan - Zonana syndrome (Liaw et al., 1997; Marsh et al., 1997, 1998a,b) and Lhermitte - Duclos disease (Liaw et al., 1997). Although each of the three conditions is characterized by distinct clinical symptoms, the affected patients share high susceptibility for benign hamartomatous tumors throughout the body early in life, as well as increased incidence of cancers of the breast, thyroid and brain (Eng, 1998).
The human PTEN gene encodes a 403 amino acid polypeptide with a high degree of homology to protein phosphatases (Li et al., 1997; Steck et al., 1997) as well as a protein associated with the actin cytoskeleton at focal adhesions, termed tensin (Lo et al., 1994). The importance of an intact PTEN phosphatase domain for its tumor suppressor function was emphasized by the findings that a majority of tumor-associated PTEN mutations map to the region encoding the phosphatase domain (Marsh et al., 1998a; Rasheed et al., 1997). Furthermore, unlike wild-type PTEN, catalytically-inactive PTEN is unable to suppress growth and tumorigenicity of PTEN-deficient glioblastoma cells (Furnari et al., 1997). PTEN is capable of dephosphorylating both phosphotyrosine and phosphoserine/threonine-containing artificial substrates in vitro (Myers et al., 1997). However, the affinity of PTEN for proteinaceous substrates is relatively low and PTEN exhibits preference for highly acidic substrates, suggesting an unusual substrate specificity. To that end, it has been shown that PTEN dephosphorylates the D3 position of phosphatidylinositol (3,4,5) trisphosphate (PI(3,4,5)P3), the primary product of phosphatidylinositol 3' kinase activity (Maehama and Dixon, 1998). Thus, PTEN activity directly antagonizes PI3'K. The relevance of PTEN PI(3,4,5)P3-phosphatase activity for its tumor suppressor function was highlighted by the fact that mutant PTEN proteins found in two unrelated patients with Cowden syndrome, as well as some of the mutant PTEN proteins found in sporadic tumors, have abrogated lipid phosphatase activity yet retain the ability to dephosphorylate a synthetic tyrosine-phosphorylated protein substrate (Furnari et al., 1998; Myers et al., 1998). PTEN has also been shown to interact directly with focal adhesion kinase (FAK) and reduce its tyrosine phosphorylation. Expression of PTEN was shown to inhibit cell migration, integrin-mediated cell spreading, and formation of focal adhesions (Tamura et al., 1998). Whether these effects are mediated via FAK or are a consequence of modulation of ILK (which is PI3'K-dependent) remains to be established.
Further mechanistic insight into the physiological function of PTEN was derived from investigations of PTEN-deficient cell lines. Immortalized mouse embryo fibroblasts (MEFs) generated from PTEN-mutant mice exhibit significantly lower sensitivity to cytotoxic stresses known to induce apoptosis, such as osmotic shock, UV-irradiation, heat treatment or stimulation with tumor necrosis factor a (Stambolic et al., 1998). Resistance to apoptotic stimuli is accompanied by constitutively elevated activity and phosphorylation of PKB, a crucial regulator of cell survival (see above). Significantly, both sensitivity to apoptotic stimuli and hyperphosphorylation of PKB in PTEN-deficient cells could be restored to wild-type levels by expression of exogenous PTEN. Examination of PI(3,4,5)P3 levels in PTEN-mutant MEFs revealed elevated intracellular levels of this lipid in comparison to that in their wild-type counterparts, in agreement with an active role of PTEN in negative regulation of PI(3,4,5)P3 levels in cells. Consistent with such a role, a number of PTEN-deficient tumor cell lines also display increased PI(3,4,5)P3 levels accompanied by hyperphosphorylation of PKB and elevated cellular survival (Dahia et al., 1999; Davies et al., 1999; Haas-Kogan et al., 1998; Li et al., 1998; Myers et al., 1998). Expression of high levels of PTEN in certain cell lines leads to apoptosis, a phenomenon that can be rescued by coexpression of activated mutants of PKB (Stambolic et al., 1998). In other cells overexpression of PTEN causes G1 arrest (Furnari et al., 1998), a potential consequence of the ability of PTEN to regulate the expression of the cell cycle regulator p27KIP1 (Sun et al., 1999). It appears that whether PTEN induces apoptosis or cell cycle arrest depends on the type of investigated cells. Resolution of this apparent discrepancy of the effect of PTEN on different cell types requires further investigation.
Mice null for PTEN die during embryogenesis between gestation day E6.5 and E9.5 (Di Cristofano et al., 1998; Podsypanina et al., 1999; Suzuki et al., 1998) from an apparent failure to form chorio-allantoic fusion (Suzuki et al., 1998). The severity of PTEN mutant phenotypes appears to depend on the genetic background, as the animals generated by three independent groups have slightly different phenotypes (Di Cristofano et al., 1998; Podsypanina et al., 1999; Suzuki et al., 1998). Mutants from at least one group gastrulate and form all three germ layers, even though they are developmentally delayed (Suzuki et al., 1998). PTEN-null embryos show abnormally patterned and expanded cephalic and caudal regions (Stambolic et al., 1998; Suzuki et al., 1998). BrdU staining has identified those regions as hyperproliferative, implicating PTEN in control of proliferation during early mouse embryogenesis (Stambolic et al., 1998). Disruption of PTEN also interferes with differentiation of embryonic stem (ES) cells into haematopoetic mesoderm (Di Cristofano et al., 1998).
Mice heterozygous for PTEN are highly susceptible to tumors. The predominant type of malignancies in PTEN+/- mice at a young age is of lymphoid origin. 15 - 20% of all mice develop thymic and peripheral lymphomas, predominantly of T-cell origin, with infiltration into multiple organs and tissues (Podsypanina et al., 1999; Suzuki et al., 1998). Moreover, gamma-irradiation decreases the time of development of thymic lymphomas in PTEN+/- mice, which was in each case accompanied by loss of heterozygosity at the PTEN locus (Suzuki et al., 1998). These tumors exhibit elevated phosphorylation of PKB in comparison to normal tissue, consistent with the notion that PTEN negatively regulates PKB signaling (Suzuki et al., 1998). Of note, v-akt, the oncogenic form of PKB, also causes mouse T cell lymphoma (AKR) (Bellacosa et al., 1991; Staal and Hartley, 1988). Lymph node hyperplasia, dysplastic intestinal polyps, thyroid neoplasms, atypical adenomatous hyperplasia in the liver, and teratocarcinoma were also observed at an increased frequency in PTEN heterozygous mice (Podsypanina et al., 1999; Suzuki et al., 1998).
Interestingly, young PTEN+/- mice fail to exhibit characteristics of patients with Cowden's, BZ and L-D syndromes. However, past six months of age, all PTEN+/- females present with atypical hyperplasia of the endometrium which in a number of cases leads to carcinoma (Podsypanina, 1999; V Stambolic et al., in preparation). Furthermore, almost all females develop breast carcinoma in situ, whereas about half of males show prostate malignancies (V Stambolic et al., in preparation). Almost all of these tumors are associated with LOH at the PTEN locus and manifest hyperphosphorylation of PKB (V Stambolic et al., in preparation), implicating PI3'K/PTEN/PKB regulated pathway(s) in the development of these tumors in mice. Thus, older PTEN heterozygous mice exhibit some of the hallmarks of PTEN-associated hamartoma syndromes, and represent a model system for their investigation in the laboratory.
The next major challenge in PTEN research is identification of modes of its regulation. It is reasonable to assume that stimuli that result in activation of PI3'K and related pathways would result in inhibition of PTEN activity. Alternatively, PTEN could be constitutively active and signals resulting in activation of PI3'K are able to transiently override the negative regulation of PI3'K-mediated pathways by PTEN. PTEN could also be regulated at the protein level by control of its expression and/or degradation. Further studies are also needed to delineate the role of PTEN in control of cell proliferation and survival. It is conceivable that the physiological function of PTEN is neither to induce apoptosis nor cell cycle arrest, but to negatively regulate the processes of cell survival and proliferation. Thus, the results of PTEN overexpression studies could represent an extreme outcome of the suppressive effects of PTEN on these processes and not a true representation of its physiological role. The balance between PI'3K, PI(3,4,5)P3, PKB and their downstream targets on one side, with PTEN on the other, functions as a molecular indicator capable of regulating the survival and/or proliferation potential of individual cells. Any alterations of this balance due to either amplifications of positive regulators of survival and proliferation, or inactivating mutations of the negative ones, can lead to tumorigenesis.
Genetics of PI3'K signaling in flies and worms
The high degree of conservation of the PI3'K pathway and its downstream mediators has allowed genetic approaches for unravelling functions (Figure 1). There are two PKB genes in the worm, C. elegans and only one in the fruit fly, D. melanogaster. In the fly, PKB is a maternal effect gene. In germ line clones (which are mutant for both the zygotic and maternal copies of the gene), loss of PKB is associated with ectopic apoptosis (Staveley et al., 1998). A similar phenotype is observed upon expression of a dominant negative mutant of PI3'K or an interfering mutant of PKB (A Manoukian, J Jing and JR Woodgeff in preparation). During normal development, certain cells undergo apoptosis. Many of these apoptotic events can be suppressed by expression of a dominantly activated PKB (PKBDD; A Manoukian, J Jing and JR Woodgeff in preparation). These data indicate that PKB activity is necessary to suppress cell death during development and that it is sufficient to block certain forms of cell death. Interestingly, the apoptotic phenotype is de-emphasized as development proceeds. Thus, interference of the PI3'K pathway at the imaginal disc stage results not in cell death but in reduction of cell size.
Genetic analysis of the PI3'K pathway is most advanced in the nematode C. elegans. In this organism the PI3'K homologue, age-1/daf-23, has been identified as one of the genes involved in regulation of overall life span. Wild-type worms live for 2 weeks at 20°C. However, under stress conditions such as food shortage or overcrowding, the animals enter a dauer stage, characterized by slowed-down metabolism, storage of fat and dormancy (reviewed in Hekimi et al., 1998; Roush, 1997; Thomas and Inoue, 1998). In this state, the worms can live up to two months. Mutations of a number of genes in the worm result in `longevity' or extension of the life span as a result of an extended dauer stage. Worms mutant for daf-2 (insulin-receptor homologue) (Kenyon et al., 1993; Kimura et al., 1997), age-1/daf-23 (PI3'K homologue) (Morris et al., 1996), pdk-1 (PDK1 homologue) (Paradis et al., 1999) and AKT-1 and AKT-2 (PKB homologues) (Paradis and Ruvkun, 1998) share a common, `longevity' phenotype, resulting from the extension of the dauer stage. daf-18, a mutant in the PTEN C. elegans homologue is able to suppress the age-1 mutant phenotype and to a lesser extent the daf-2 phenotype, providing genetic evidence that PTEN acts as a negative regulator of PI3'K-regulated pathways (Gil et al., 1999; Mihaylova et al., 1999; Ogg and Ruvkun, 1998; Rouault et al., 1999). Mutations of another gene, daf-16 (Lin et al., 1997; Ogg et al., 1997), are also able to suppress daf-2, age-1/daf-23, pdk-1 and akt-1 and akt-2 phenotypes. As mentioned above, daf-16 encodes a homologue of the mammalian transcription factors FKHR, AFX and FKHRL1, which have recently been shown to be phosphorylated and negatively regulated by mammalian PKB (Figure 1). Thus, a pathway homologous to the insulin signaling pathway in mammals, dates back 700 - 800 million years, prior to divergence of nematodes.
The nematode studies have already played a key role in identifying pertinent PKB targets and it is likely that further substrates will be revealed by suppressor and enhancer screens in this and other organisms.
Pros and cons of apoptosis signaling
Since activation of PI3'K provides a survival signal, it is a prime target for regulation by pro-apoptotic pathways. Evidence for such cross-talk has come from studies of the inhibitory effects of ceramides on PI3'K and PKB activation (Summers et al., 1998; Zhou et al., 1998; Zundel and Giaccia, 1998). Ceramides can be produced by certain pro-inflammatory cytokines such as TNF. In many cells, this cytokine induces divergent signals which promote (via caspase induction) as well as suppress (via NF-B and SAPK/JNK activation) cell death (Basu and Kolesnick, 1998). TNF has also been reported to activate PKB in HeLa cells (Pastorino et al., 1999). This cytokine thus acts as a double-edged sword, sensitizing cells and forcing a decision on their fate (Baker and Reddy, 1998). Interactions have also been reported with other signaling pathways such as the ERK and p38 MAPK systems, resulting in modulation (Hayashi et al., 1999). The intimate interaction between pro- and anti-apoptotic signals is perhaps a reflection of the dangers associated with suppression of cell death, as evidenced by PTEN mutations.
Overview, potential therapies, new targets, etc.
Multiple components of PI3'K-regulated cellular pathways are present in most cells of the body and it is reasonable to assume, given the multitude of ways in which PI3'K is regulated, that their activation will depend on cell type and its immediate microenvironment. The ability of PI3'K to regulate a variety of cellular processes also suggests that the cellular context of downstream targets of this pathway could represents a determining factor in the interpretation of a PI3'K-generated signal. PI3'K pathway seems to be conserved throughout eukaryotic evolution, judged by recent discovery of C. elegans PKB, PDK1 and PTEN and their interplay with previously characterized PI3'K and insulin receptor tyrosine kinase homologues in this organism.
Identification of oncogenic forms of PI3'K and PKB, together with a high rate of PTEN mutations in a variety of malignancies, has established PI3'K signaling as one of the most frequently deregulated cellular pathways in human cancer. Antibodies specific for the phosphorylated forms PKB have greatly facilitated monitoring of the status of this enzyme and the PI3'K pathway in general. Similar to phospho-specific MAP kinase antibodies, these reagents have allowed facile determination of the flux through the PI3'K pathway in cells and tissue sections and will undoubtedly aid in assessment of the status of this anti-apoptotic cascade in various pathologies. In view of its relevance to human cancer, the PI3'K pathway is receiving much attention from researchers focused on the development of novel anti-cancer therapies. Known PI3'K inhibitors, such as wortmannin and LY294002, are highly toxic and exemplify some of the problems associated with therapies targeting this molecule. However, therapies aimed at a target further downstream in this pathway, might alleviate some of the broader side effects a compound targeting an upstream component might have (indeed wortmannin and LY294002 also inhibit protein kinases such as ATM and DNA-PK; Sarkaria et al., 1998; Wymann et al., 1996). Since activation of PI3'K helps tumor cells tolerate the consequences of genomic instability, it is possible that tumor cells will exhibit differential sensitivity to inhibitors of PKB, its targets and its regulators, providing a clinically useful therapeutic index. Indeed, while tumor cell activation of the pathway reduces the efficacy of conventional chemotherapeutics and irradiation treaments, dependence upon chronic PI3'K signaling may prove to be an achilles heel.
TW Mak and JR Woodgett are supported by grants from the Medical Research Council and Terry Fox Foundation for Cancer Research. JR Woodgett is additionally supported by a Howard Hughes International Scholarship.
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Figure 1 Components of the PI3'K signaling pathway in mammals and C. elegans. Components of the PI3'K pathway have been remarkably conserved between multicellular organisms allowing genetic analysis and identification of new components. The worm genes and their corresponding mammalian counterparts are illustrated
Table 1 Tumors associated with dysregulation of the PI3'K pathway
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