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The PTEN–PI3K pathway: of feedbacks and cross-talks

Abstract

The tumor suppressor PTEN was originally identified as a negative regulator of the phosphoinositide 3-kinase (PI3K) signaling, a main regulator of cell growth, metabolism and survival. Yet this function of PTEN is extremely relevant for its tumor-suppressive ability, albeit the recent characterization of many PI3K-independent tumor-suppressive activities. PI3K-mediated PIP3 production leads to the activation of the canonical AKT-mTORC1 pathway. The implications of this signaling cascade in health and disease have been underscored by the high number of regulators within the pathway whose alterations give rise to different malignancies, including familiar syndromes, metabolic dysfunctions and cancer. Moreover, PI3K is tightly buffered at multiple levels by downstream components, which have turned this signaling pathway literally upside down. PI3K and its downstream components in turn cross-talk with a number of other pathways, thereby leading to a complex network of signals that may have dramatic consequences when perturbed. Here, we review the current status of the PTEN–PI3K signaling pathway with special emphasis on the most recent data on targets and regulation of the PTEN–PI3K axis. This provides novel provocative therapeutic implications based on the targeted modulation of PI3K-cross-talking signals.

Components of the PI3K pathway

Phosphoinositide 3-kinases (PI3Ks) are a family of proteins involved in the regulation of cell growth, metabolism, proliferation, glucose homeostasis and vesicle trafficking (Engelman et al., 2006). Most of the members of this family are bound to regulatory subunits, which determine the signals modulating its activity. There are three members in the family (Cantley, 2002): class I PI3K, which is divided into IA and IB, is activated by receptor tyrosine kinases (RTKs, PI3K1A) and G-protein-coupled receptors (GPCRs, PI3K1B). Class IA and IB PI3Ks have different regulatory subunits, p85α/p85β/p55 for IA and p101/p84/p87PIKAP for IB. This class is characterized for generating primarily PI-3, 4.5-P3 (PIP3) (Katso et al., 2001).

PI3K class II utilizes in vitro PI-3-P to generate PI-3, 4-P2 and can also produce PI-3-P from PI. This class does not require a regulatory subunit to function, and comprises three different isoforms (α, β and γ) that diverge in the N terminus and present different domains within the C terminus. Class II PI3K is involved in membrane trafficking and receptor internalization and can be activated in response to RTKs, integrins and cytokine receptors (Engelman et al., 2006).

Class III PI3K (Vps34), which was first identified in the budding yeast (Herman and Emr, 1990), is involved in vesicle trafficking (Backer, 2008) and cross-talks with class I PI3K through the regulation of mTORC1 signaling, as we will discuss later.

Class I PI3K is the most studied among the three members of the family. For the purpose of this review, we will focus mostly on this class, referring to it as PI3K unless otherwise specified.

PTEN

The Phosphatase and Tensin homolog deleted on chromosome TEN was originally discovered as a candidate tumor suppressor mutated and lost in various cancers (Li and Sun, 1997; Li et al., 1997; Steck et al., 1997). Several lines of evidence soon highlighted PTEN as a lipid phosphatase-hydrolysing phosphates in position 3′ from phosphoinositides (Maehama and Dixon, 1998; Stambolic et al., 1998). The major function of PTEN is the buffering of PI3K signaling; yet recent studies point to additional novel, lipid phosphatase-independent functions that may contribute to its tumor-suppressive activity (Carracedo et al., 2008; Salmena et al., 2008). The loss and mutation of PTEN in various cancers lead to hyperactive PI3K signaling. For example, PTEN is commonly mutated in its phosphatase domain (Eng, 2003); and in glioblastoma, mutations that impair its proper membrane localization might result in deficient tumor-suppressive activity (Walker et al., 2004). It is therefore clear that PTEN is a main player in the regulation of PI3K signaling and, as we will discuss, perturbations in its levels or function can dramatically impact on this pathway.

PIP3-dependent pathways

Upon 3′ phosphorylation of PI-4,5-P2 by PI3K, proteins containing pleckstrin homology (PH) and PH-like domains are recruited to the plasma membrane, thereby transmitting the signal elicited by PI3K activation (Engelman et al., 2006). One of the best-characterized members of this group of proteins is the pro-survival AKT kinase. AKT contains a PH domain; upon PIP3 production it anchorages to the membrane, where another phosphoinositide-binding protein, PDK1 (Alessi et al., 1997), and a recently discovered protein complex, mTORC2 (Sarbassov et al., 2004, 2005), phosphorylate and activate the kinase. Activated AKT mediates several of the well-described PI3K responses, mainly growth, metabolism, survival and glucose homeostasis (Manning and Cantley, 2007). Therefore, the PI3K-AKT axis is considered the canonical PI3K signaling. But, as we will discuss later, PI3K leads to the modulation of other pathways that are of great importance for the described function of this kinase.

AKT phosphorylates up to 100 substrates thereby modulating a variety of cellular functions. First, AKT signaling exerts a strong antiapoptotic effect through the phosphorylation and inhibition of key pro-apoptotic proteins, such as BAD, MDM2 and members of the Forkhead family (del Peso et al., 1997; Biggs et al., 1999; Brunet et al., 1999; Guo et al., 1999; Kops et al., 1999; Nakae et al., 1999; Rena et al., 1999; Takaishi et al., 1999; Tang et al., 1999; Mayo and Donner, 2001; Ashcroft et al., 2002). Second, AKT activates cell proliferation by inactivating p27 (Fujita et al., 2002) and inhibiting glycogen synthase kinase 3 (GSK3)-mediated Myc and cyclin D1 inhibition (Vivanco and Sawyers, 2002). Third, this kinase regulates a subset of proteins involved in growth, metabolism and angiogenesis. AKT phosphorylates and inactivates GSK3β (Cross et al., 1995), increases glucose transporter Glut4 translocation to plasma membrane by blocking AS160 (Sano et al., 2003) and, through FOXO inactivation, inhibits phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (Burgering, 2008). All these actions converge in an increased glucose catabolism rate. Additionally, AKT is one of the main regulators of a complex involved in protein translation and ribosome biogenesis; this is mTORC1, which is composed of the protein kinase mTOR and a series of interactors (Guertin and Sabatini, 2007). AKT regulates this complex through the phosphorylation of two different substrates. On the one hand, this kinase phosphorylates and inactivates the GTPase-activating protein (GAP) TSC2 (Dan et al., 2002; Inoki et al., 2002; Potter et al., 2002), which forms a complex with TSC1 to inhibit a small GTPase, Rheb (Ras-homolog enriched in brain) (Garami et al., 2003; Inoki et al., 2003; Tee et al., 2003; Zhang et al., 2003b). Rheb activates mTORC1 through the inhibition of the FKBP38, a novel negative regulator of mTORC1 (Bai et al., 2007). On the other hand, AKT phosphorylates and inhibits PRAS40 (proline-rich AKT substrate of 40 KDa), which negatively regulates the activity of mTORC1 through the competition with the GTPase Rheb (Kovacina et al., 2003; Sancak et al., 2007; Vander Haar et al., 2007). Through this dual regulation, AKT promotes the activation of mTORC1 pathway, which turns on the translational machinery to produce ribosomes and increases the protein synthesis rate.

Of note, the production of PIP3 modulates a different branch of signals, which will not be discussed in depth in this review.

mTORC1 pathway

As aforementioned, a main part of PI3K signaling converges in mTORC1 activation. mTORC1 phosphorylates and activates S6 kinases 1 and 2 (Guertin and Sabatini, 2007), which in turn activates protein translation and ribosome biogenesis through phosphorylation of different substrates in addition to ribosomal protein S6, including PDCD4, eEF2 kinase and eIF4B (Wang et al., 2001; Raught et al., 2004; Dorrello et al., 2006). On the other hand, mTORC1 phosphorylates and inactivates 4E-binding protein 1 (4EBP1), thereby releasing the inhibition of the eukaryotic initiation factor 4E (eIF4E) (Guertin and Sabatini, 2007). Taken together, mTORC1 modulates a variety of substrates, which ultimately upregulate protein synthesis. This pathway has turned out to be enormously interesting for the treatment of several diseases due to the discovery of a specific inhibitor, rapamycin, a macrolide extracted from a fungi Streptomyces hygroscopicus (Vezina et al., 1975). Rapamycin binds an intracellular protein, FKBP12, which blocks mTORC1 activity through mechanisms not yet well understood (Sabatini et al., 1994). The ability of rapamycin to inhibit mTORC1, together with the observation that this complex is hyperactive in many cancers, has promoted a number of clinical trials for the treatment of a variety of cancers. Meanwhile, the benefit of mTORC1 inhibition for the treatment of many cancers remains to be established (Guertin and Sabatini, 2007).

The importance of mTOR in the response to PI3K activation goes beyond mTORC1 activation. Recently, mTOR was found to interact with a different subset of proteins in a complex called as mTORC2 (Sarbassov et al., 2004, 2005). This complex is now accepted as the factor responsible for AKT phosphorylation in serine 473 (Ser473), thus locating mTOR both at the top and at the bottom of this pathway.

PI3K pathway in disease

Since its discovery, the role of PI3K pathway in disease has been highlighted by the number of members of the pathway whose aberrant function associates with the development of malignancies (Engelman et al., 2006). As this pathway is a central integrator of metabolism and survival/growth signals, it is not surprising that dysfunctional PI3K cascade would lead to metabolic diseases and cancer.

With respect to metabolism, many studies in mouse models and the further support of clinical data have made it evident that reduced PI3K activity can play a role in insulin sensitivity and non-insulin-dependent diabetes mellitus (type II diabetes). Data coming from mice lacking p110α regulatory subunits have shed light on the dual role of p85 in PI3K regulation in response to insulin (Katso et al., 2001). Heterozygous deletion of either p85α or β in mice leads, surprisingly, to increased sensitivity to insulin, whereas compound heterozygous mutants fail to activate PI3K in response to insulin. The explanation for this dual effect is based on the fact that p85 can buffer the binding of p110α/p85 complex when expressed in excess within the cell. Therefore, partial p85 reduction would favor PI3K activation by reducing insulin receptor substrate-1 (IRS-1)-bound p110α-free p85, whereas a further decrease would attenuate p110α activation through disruption of p110α/p85 complexes (Luo et al., 2005a). In support of a role for PI3K in diabetes, p110α and β double heterozygous mutant mice show glucose intolerance. On the other hand, Pten loss of function exhibits dramatic consequences for metabolism homeostasis. As an example, Pten-deleted muscle, liver or fat tissue exhibits increased glucose sensitivity (Stiles et al., 2004; Kurlawalla-Martinez et al., 2005; Wijesekara et al., 2005).

The recent discovery of a role for class 3 PI3K (VPS34) in amino-acid sensing might similarly provide a new angle for the role of this PI3K in metabolism. VPS34 was originally involved in vesicle trafficking (Herman and Emr, 1990). Further studies proposed a role for this kinase in the formation of autophagosomes, double-membrane organelles involved in the digestion of intracellular components in response to starvation and stress (Backer, 2008). The role of VPS34 in autophagy already implicates this kinase in aging and response to nutrient deprivation. But, recently, a novel function for VPS34 in amino-acid sensing was discovered by the group of Drs Thomas and Kozma. In an elegant study, they found that the ability of mTORC1 to sense amino acid was not dependent on the class 1 PI3K, but on the class 3 (Nobukuni et al., 2005). A more recent study by this group reveals the mechanism of how VPS34 senses amino acids, a phenomenon that depends on the release on calcium from the endoplasmic reticulum to the cytoplasm (Gulati et al., 2008). Therefore, this finding suggest that aberrant Class 3 PI3K signaling could have a deep impact in proper amino-acid sensing by mTOR, with possible implications in metabolic processes.

The evidence of a role for this pathway in diabetes is underscored in humans by the presence of genetic alterations of PI3K regulators in patients with this disease. p85 levels are found increased in some patients with insulin resistance (Bandyopadhyay et al., 2005) Moreover, polymorphism of PI3KR1 (which encodes for p85) together with those found in IRS-1 and IRS-2 has been associated with diabetes (Almind et al., 1996; Kawanishi et al., 1997; D'Alfonso et al., 2003; Esposito et al., 2003). On the other hand, AKT2 has been found to be mutated in familiar insulin resistance (George et al., 2004), thereby enforcing the concept that PI3K, through modulation of targets involved in metabolic regulation, fine-tunes glucose metabolism in response to extracellular stimuli.

Alterations in PI3K pathway have been largely associated with cancer as well as different cancer susceptibility syndromes. From the top of the pathway, PI3K class IA is mutated and amplified in a variety of cancers (Samuels et al., 2004). The most frequently activating mutations (Hot Spots) in PI3K are located within the kinase domain, H1074, and in the helical domain, E542-5. Although these two mutations may have different impacts on PI3K targets, both drive transformation in vitro (reviewed in Zhao and Vogt, 2008). Interestingly, there appears to be a tissue-specific preference for mutation or amplification of PI3K. Although amplifications but not mutations are frequent in ovarian and gastric tumors, mutations in the absence of amplifications of this gene often occur in breast and colorectal cancer (Bader et al., 2005). Of interest, somatic mutations in PIK3R1 have been found in ovarian and colon cancer, and in fact partial reduction of p85 levels enforces the tumorigenesis by heterozygous Pten loss in mice (Luo et al., 2005b), providing support to the hypothesis that alteration of PI3K signaling at multiple levels can result in cancer (Bader et al., 2005). Of note, although the implications of PI3K1A mutation and amplification are widely characterized, the role of other PI3K isoforms is still unclear. Indeed, according to recent studies different PI3K isoforms would require distinct signaling pathways to drive transformation (Denley et al., 2008), which in turn further highlights the relevance of cross-talks involving PI3K and other pathways for tumor progression.

The relevance of PTEN in cancer arises from its discovery, when three independent groups characterized a gene located in chromosome 10 as highly mutated and lost in several cancers (Li and Sun, 1997; Li et al., 1997; Steck et al., 1997). PTEN is a lipid phosphatase with a lipid-binding domain that allows anchorage to the plasma membrane. Although described as a highly stable protein, different groups have recently reported PTEN is tightly regulated at the transcriptional level, as well as by post-translational modifications (Salmena et al., 2008). Mutations either in the catalytic domain, C2 domain (Eng, 2003) or in ubiquitinylation sites (Trotman et al., 2007) have been shown to correlate with the development and progression of cancer. Moreover, germline heterozygous alterations in PTEN locus are associated with the development of a group of syndromes generally referred as PTEN hamartoma tumor syndromes, which includes Cowden, Lhermitte–Duclos, Bannayan–Riley–Rubalcaba, Proteus and Proteus-like syndromes (Eng, 2003). This disease is characterized by the presence of developmental and neurological defects, hamartomas and cancer susceptibility. Mouse models of Pten loss have shown that deletion of one allele of Pten results in lethal autoimmune disorders, and various forms of epithelial cancers (Di Cristofano et al., 1998, 1999). These data have led to the notion that, at least in the mouse, Pten is haploinsufficient for its tumor-suppressive activity.

Because of the lethal nature of complete Pten loss, several groups have addressed the effect of the acute loss of this tumor suppressor through tissue-specific Pten-conditional deletion. Surprisingly, complete Pten deletion does not have the pro-tumorigenic action predicted. Indeed, acute Pten loss in the hematopoietic stem cell compartment leads to exhaust of the hematopoietic stem cell (Yilmaz et al., 2006; Zhang et al., 2006b) and acute Pten loss in the prostate leads to a strong p53-dependent senescence response that opposes cancer progression (Chen et al., 2005). On the other hand, Pten deletion in neural cells increases the size of the brain and perturbs its function without leading to cancer. At the cellular level, Pten loss induces glial cell proliferation but does not have any effect on neurons (Fraser et al., 2004, 2008; Endersby and Baker, 2008). These studies suggest that tumors may not select for a complete loss of function of PTEN in the initial steps of tumorigenesis (as happens in prostate cancer, where approximately 70% of the tumors have heterozygous alterations in PTEN at presentation and lose the other allele at later stages), which may have relevance in the understanding of the ‘just-right’ dose (Albuquerque et al., 2002) of PI3K that would be of advantage for cancer to progress.

The relevance of AKT in the transduction of PI3K signals suggests that the activity of this kinase could be altered through mutations in AKT genes. Indeed, myristoylated constitutively active Akt1 promotes aberrant prostate growth (prostate intraepithelial neoplasia) in transgenic mice where the gene is set under prostate-specific promoter regulation (Majumder et al., 2003). Interestingly, Akt overexpression leads to an mTORC1-dependent (rapamycin-sensitive) glucose metabolism signature, which underscores the major role of mTORC1 in AKT-driven tumorigenesis (Majumder et al., 2004).

The role of AKT in human cancers was for a long time thought to be restricted to AKT (AKT1 and 2) gene amplification, as found in gastric, ovarian, pancreas and stomach cancers (Engelman et al., 2006). But recently, the group of Thomas reported a novel point mutation in the lipid-binding pocket of AKT1 in breast, colorectal and ovarian cancer patients that leads to AKT1 targeting to plasma membrane and activation of downstream effectors (Carpten et al., 2007). Mutant Akt1 is able induce leukemia in mice and recent studies have reported the same mutation in a subset of squamous cell lung carcinoma and breast cancer (Kim et al., 2008; Malanga et al., 2008).

Lastly, alterations in TSC genes (TSC1 and TSC2) result in the development of an autosomal dominant disease, tuberous sclerosis, characterized hamartomas in brain, heart, lung and kidney, neurological disorders, angiomyolipomas and rhabdomyomas (Jozwiak et al., 2008). Moreover, most of the patients who develop these malignancies (with the exception of the cerebral tubers) exhibit loss of the intact allele, a compelling example of a tumor suppressor that conforms to the ‘two-hit hypothesis’ for tumor suppression proposed by Knudson (Knudson, 1996).

Upstream regulators of PI3K signaling

As mentioned above, PI3K is activated mainly upon nutrient and growth factor stimulation. The regulatory subunit of PI3K1α, p85 binds to tyrosine-phosphorylated proteins, typically receptor tyrosin kinases and adaptor proteins, to activate PI3K (Cantley, 2002). In a prototypical example, the engagement of the IRS protein by the tyrosine kinase receptors leads to the binding of the p85–PI3K1A heterodimer, which mediates the transmission of the extracellular signal (that is, insulin) to the interior of the cell. RTK can also activate PI3K through the engagement of Ras and consequential binding of the GTPase to the Ras-binding domain (RBD) of PI3K (Engelman et al., 2006). There are other mechanisms of PI3K activation by extracellular signals. Another family of plasma membrane receptors, the GPCRs, can activate some PI3K isoforms directly (Schwindinger and Robishaw, 2001), which is thought to occur through the βγ subunits of activated heterotrimeric G proteins and indirectly (Schafer et al., 2004) through transactivation of RTK.

The intensity of PI3K activation is negatively regulated by a number of proteins. The main negative regulator is the phosphatase PTEN. As aforementioned, PTEN dephosphorylates phosphoinositides in position 3′, being therefore in charge of the termination of the signal transmitted by PI3K. The levels of PTEN are controlled at the transcriptional level by PI3K. Through the regulation of the transcription factor NF-κB (Vasudevan et al., 2004), PPARβ/δ agonists (Han et al., 2008) and TNFα (Kim et al., 2004) repress the expression of PTEN. This negative regulation is of importance in lung cancer cells, where downregulation of PTEN by PPARs promotes cancer cell proliferation. On the other hand, ubiquitinylation-dependent proteasomal degradation of PTEN reduces its levels (Torres and Pulido, 2001; Wu et al., 2003; Wang et al., 2007). To date, NEDD4-1 is the first and only identified E3 ligase for PTEN (Wang et al., 2007). Interestingly, NEDD4-1 transcription is positively regulated by PI3K pathway (Ahn et al., 2008), therefore representing a positive feedback for PTEN degradation and PI3K activation. Although the biological conditions where NEDD4-1 would ubiquitinylate PTEN are unclear, the existence of this or other E3 ligases targeting PTEN would represent potential targets for cancer therapy in tumors where PTEN is downregulated.

The activity of PTEN is also controlled by the PI3K pathway. The loss of the regulatory subunit of PI3K1A, p85, results in the decrease of Pten activity in p85 liver conditional knockout mice (Barber et al., 2006). Similarly, δ-isoform of PI3K has been shown to regulate PTEN activity through a RhoA-ROCK-dependent signaling, therefore impacting on chemotaxis and proliferation of macrophages (Papakonstanti et al., 2007). Lastly, PTEN nuclear–cytoplasmic shuttling is of great importance for the proper tumor-suppressive activity of the phosphatase (Denning et al., 2007; Trotman et al., 2007). NEDD4-1-mediated PTEN monoubiquitinylation has been defined as the key for PTEN to shuttle to the nucleus (Trotman et al., 2007). Similarly, activated PI3K promotes PTEN nuclear exclusion through a chromosome region maintenance 1/S6K-dependent mechanism (Liu et al., 2007). The effect of aberrant PTEN localization in PI3K signaling is not yet completely understood. Although forced nuclear PTEN expression can reduce AKT phosphorylation (Trotman et al., 2007), it has also been proposed that PTEN is less efficient in hydrolysing nuclear PIP3 (Lindsay et al., 2006), and could have other functions that do not depend on dephosphorylating phosphoinositides (Salmena et al., 2008).

Downstream PI3K, the activation of AKT is also negatively regulated by different proteins. On the one hand, protein phosphatase (PP) 2A dephosphorylates primarily Thr308, whereas the phosphatase PHLPP dephosphorylates Ser473 (Bayascas and Alessi, 2005; Gao et al., 2005). On the other hand, a pseudokinase, namely Tribbles homolog 3, binds to and inhibits AKT, which leads to insulin resistance (Du et al., 2003).

Recently, a well-characterized tumor suppressor, the promyelocytic leukemia (PML) protein was uncovered as a key regulator of the PI3K at multiple levels. In one study, it was found that Pml regulates PP2A activity toward nuclear AKT residing in the nuclear bodies, which may account for the acceleration observed in tumors Pten heterozygous/Pml-defective mice (Trotman et al., 2006). In an independent study, Pml loss was also shown to promote neo-angiogenesis in femoral arterial ligation experiments. The mechanism of this phenomenon depends on the sequestration of mTOR to the nuclear bodies in hypoxic conditions, which accounts for the reduction in hypoxia-inducible factor 1α translation and ribosomal protein S6 phosphorylation (Bernardi et al., 2006).

Feedback regulators of PI3K signaling

In the past years, several reports have described that PI3K is tightly controlled by downstream components of the pathway, thus providing feedback regulation in response to extracellular signals (Figure 1).

Figure 1
figure1

Feedback regulation of the PTEN–PI3K pathway. The canonical PTEN–PI3K pathway (green) is regulated at multiple levels. First, membrane receptor (RTK and GPCR) and its adaptors activate PI3K isoforms leading to the transmission of the signal to the interior of the cell (blue). PI3K signal intensity is controlled by a number of proteins, including, but not limited to PTEN and TSC1/2. Moreover, PI3K activation activates several feedback loops (rose), some of them promote further pathway activation (TSC1/2–mTORC2, NEDD4-1–PTEN, PI3K–RhoA–ROCK–PTEN) and some of them exert a negative effect on it (multiple signals from S6K, eIF4E—NBS-1–PI3K, FOXO–PP2A–AKT). GPCR, G-protein-coupled receptor; RTK, receptor tyrosine kinase; PDGFR, platelet-derived growth factor receptor; PI3K, phosphoinositide 3-kinase; NBS-1, Nijmegen breakage syndrome 1; UPR, unfolded protein response.

The most prominent negative feedback regulating PI3K pathway is the one triggered downstream of mTORC1. The first observation of the ability of PI3K downstream components to regulate PI3K activation arose from a series of studies showing that chronic insulin stimulation led to the phosphorylation and proteasomal degradation of the adaptor protein IRS-1 (Haruta et al., 2000; Tremblay and Marette, 2001; Berg et al., 2002; Hartley and Cooper, 2002; Zhande et al., 2002; Greene et al., 2003; Pirola et al., 2003). Since then, a number of groups have shown that insulin-mediated PI3K activation can lead to the downregulation of IRS-1 activation through transcriptional regulation and post-translational modifications. Further studies showed that loss of TSC complex resulted in impaired activation of AKT upon insulin (Harrington et al., 2004; Shah et al., 2004). The analysis of the gene expression profile in Tsc2-expressing versus -defective cells unraveled that Tsc1/2 loss results in a strong repression of IRS-1 transcription (Harrington et al., 2004). The mechanism of this transcriptional repression depends on the activation of mTORC1 signaling, as rapamycin treatment and silencing of S6K genes restores IRS-1 levels. Moreover, as mentioned above, TSC1 or TSC2 loss in human patients results mostly in benign tumors. Our group, along with the Cantley lab, recently demonstrated that Tsc2 heterozygous mice develop malignancies in which this feedback inhibition is overactive (Ma et al., 2005b; Manning et al., 2005). Indeed, PI3K activation induced by heterozygous Pten deletion is able to accelerate the progression of Tsc2 loss-derived malignancies (Manning et al., 2005). This suggests that one of the mechanisms by which Tsc loss would not result in more aggressive tumors is based on the strong brake provided by the negative feedback signaling at the very top of PI3K pathway, to the extent that forced PI3K activation induced by heterozygous Pten deletion is able to accelerate the progression of Tsc2 loss-derived malignancies (Manning et al., 2005). Similarly, in the fly, Radimerski et al. (2002) showed that dS6K reduction could restore PI3K activation status and rescue larval lethality induced by upon Tsc1/2 loss.

Loss of function of the TSC complex has also been shown to impact RTK levels. Studies from the group of Dr Kwiatkowski demonstrated that mTORC1 hyperactivation by either Tsc2 or Pten loss, as well as by PI3K activation, leads to the repression of PDGFRA and B transcription, which impacts not only in PDGF signaling to AKT but also on the proper transmission of the signal from other growth factor receptors (Zhang et al., 2003a, 2007). Overall, these data provide support for the concept that this negative regulation of PI3K by downstream components may be of great importance in cell physiology, as well as in pathological conditions (Manning, 2004).

Although IRS-1 transcriptional repression might be a critical component of the negative feedback upon chronic PI3K activation, Harrington et al. (2004) showed in the same study that a downstream target of mTORC1, S6K1 was able to phosphorylate and inhibit IRS-1 in Ser302. This phosphorylation of IRS-1 is thought to inhibit its activity, preventing its recruitment and binding to RTKs. Another study has recently suggested that in conditions of obesity, as in db/db mice (in which a mutation in the cytoplasmic domain of the long form of the leptin receptor, Ob-Rb, results in loss of expression of this isoform and therefore alters STAT3 signaling in satiety response; Chen et al., 1996; Ghilardi et al., 1996), S6K can phosphorylate IRS-1 on a different residue, Ser1101, thus reducing its function and leading to insulin resistance in vivo in skeletal muscle (Tremblay et al., 2007). mTOR kinase itself might be able to phosphorylate this adaptor protein on Ser636/639 in certain circumstances (Ozes et al., 2001; Tzatsos and Kandror, 2006), and other key regulators of this pathway can similarly modulate this adaptor protein (Jakobsen et al., 2001). Therefore, both transcriptional repression and inhibitory phosphorylation of IRS-1 by downstream components account for the feedback inhibition of PI3K.

The concept that mTORC1 hyperactivation would result in the inhibition of PI3K also raised the question of whether mTORC1 inhibitors would have the undesirable effect of activating PI3K. Although previous studies had shown that the consequence of functional loss of the TSC complex on PI3K was prevented by rapamycin (Harrington et al., 2004), it was not until 2006 that the group of Dr Rosen demonstrated that rapamycin treatment induced AKT activation in cancer patients, a strong drawback for the use of this family of inhibitors as single agent in the clinic (O'Reilly et al., 2006). In line with this idea, many groups have subsequently proved in different types of human cancers that mTORC1 inhibition can lead to PI3K activation and therefore proposed patient stratification for the utilization of mTORC1 inhibitors and dose adjustment to improve the clinical outcome (Baselga et al., 2008; Cloughesy et al., 2008).

Although the feedback signaling to AKT activation is widely accepted and provides an explanation to the unexpected poor results with rapamycin in the treatment of certain cancers, the effect of mTORC1 inhibitors on other pathways has not been yet achieved. To answer this question, our group recently investigated the effect of mTORC1 inhibition on a pro-survival pathway other than PI3K in patients subjected to RAD001 treatment. We surprisingly found that RAD001 treatment induces a profound activation of the ERK–MAPK pathway. Furthermore, we find that this activation relies on a negative feedback involving S6K—IRS-1–PI3K–RAS and that the combination of MEK and mTORC1 inhibitors already being tested in clinical trials provides a more efficient antitumoral effect in vitro as well as in vivo (Carracedo et al., 2008). This study therefore provides the basis for further patient stratification and the rationale for the combination of this class of inhibitors in the treatment of cancer.

Although the S6K-IRS-1 negative feedback might be the most prominent mechanism leading to PI3K activation, other negative feedbacks have also been proposed. mTORC1 inhibition in rhabdomyosarcoma cells leads to AKT activation through a S6K–IRS-1-independent signal that relies on IGF1R activation (Wan et al., 2007). Along the same line, acute myeloid leukemia cells upregulate IGF1 and IRS-2, but not IRS-1, upon RAD001 treatment (Tamburini et al., 2008). Another study proposes that eIF4E activation leads to AKT activation through a Nijmegen breakage syndrome 1-PI3K-dependent mechanism (Culjkovic et al., 2008) and conversely, PI3K has been proposed to signal directly to eIF4E upon rapamycin treatment (Sun et al., 2005). Downstream PI3K, members of the FOXO family can inhibit PP2A phosphatase, thereby increasing AKT phosphorylation (Ni et al., 2007), and activated S6K can overcome the effect of AKT inactivation through direct phosphorylation of GSK3 (Zhang et al., 2006a). In the context of Tsc1/2 loss, Ozcan et al. (2008) recently showed that mTORC1 hyperactivation leads to aberrant protein synthesis rate and activation of the unfolded protein response. Importantly, the activation of this pathway impacts in IRS-1 phosphorylation status, therefore reducing the ability of extracellular signals to activate PI3K. Finally, a recent study by the Manning group showed that the TSC complex, binds to and regulates mTORC2 activity, therefore promoting an mTORC1 ON/mTORC2 OFF versus an mTORC1 OFF/mTORC2 ON status (Huang et al., 2008).

Overall, the mechanisms developed by the cell to sense the intensity of PI3K activation by extracellular signals are complex and involve different pathways, and may vary from tissue to tissue. Whereas in physiological conditions the proper functioning of this feedback might be of importance for the metabolic processes, re-activation of this brake in conditions of inhibition of mTORC1 could represent a very attractive therapeutic target to oppose tumor progression (Lopiccolo et al., 2007).

Cross-talk of PI3K and other signaling pathways

Regulation of other signaling cascades by PI3K pathway

The PI3K pathway is not limited to the regulation of the canonical AKT-mTORC1 cascade. Indeed, PI3K can modulate the activation status of other pathways (Figure 2). PI3K pathway impacts on Ras–MAPK at multiple levels. Ras activation by PI3K has been well characterized in (lysophosphatidic acid) LPA-induced MAPK activation, where PI3K pharmacological inhibitors blunt the activation of ERK kinase. This activation partially relies on Gβγ subunits and Shc-Grb2-Sos activation as well as in PIP3-mediated PKCζ activation (Yart et al., 2002). Nevertheless, the specific requirement of PI3K for Ras activation might be tissue-specific and involve additional signaling molecules (Yart et al., 2002). Similarly, somatostatin receptor (Lahlou et al., 2003), as well as insulin and low doses of EGF (Wennstrom and Downward, 1999) can drive Ras activation through PI3K, which may be due to the ability of PIP3 to recruit GAP/Shp2 in some circumstances (Yart et al., 2001; Sampaio et al., 2008). On the other hand, some PI3K inhibitors such as the cytokine β-galactosidase-binding protein (βGBP) inhibit MAPK (Wells et al., 2007). The cellular context in which PI3K signals to Ras is not yet elucidated, and further research on the role of PI3K-Ras binding, PI species production and involvement of the different PI3K isoforms is required for the proper delineation of this signaling cross-talk. Similarly, the biological consequences of Ras activation by PI3K need to be investigated as they may selectively impact in some cellular responses but not others.

Figure 2
figure2

Pathways regulated by PI3K. The cascades modulated upon PI3K activation go beyond the canonical PI3K pathway (green). Several members of this pathway control the activation status of the Ras-MAPK pathway (PTEN, PI3K, PDK1, AKT, Rheb). On the other hand, PI3K and AKT also regulate other pathways (ASK1–JNK, Rac). The pathways under the control of PI3K pathway are shown in blue. PI3K, phosphoinositide 3-kinase; Rheb, Ras-homolog enriched in brain.

PI3K pathway can directly modulate the Ras target Raf therefore bypassing the GTPase. AKT phosphorylates c-Raf in Ser259 (Zimmermann and Moelling, 1999) and B-Raf (Guan et al., 2000). This inhibitory phosphorylation on Raf reduces the activity of MAPK pathway (Zimmermann and Moelling, 1999). This action of AKT on MAPK pathway is crucial for proper regulation of differentiation in some tissues and for cell survival (Rommel et al., 1999; Reusch et al., 2001; Mabuchi et al., 2002; Lee et al., 2008) and may as well impact on the response to growth factors (Guan et al., 2000). Another protein kinase with similar catalytic domain to AKT, SGK has also been suggested to phosphorylate Raf in an AKT site (Zhang et al., 2001). In which circumstances AKT inhibits Raf is still unclear; and the strength of PI3K activation may regulate this cross-talk, as AKT phosphorylation of Raf is more prominent at higher PI3K activity (Moelling et al., 2002).

The small GTPase Rheb also regulates Raf (Yee and Worley, 1997; Im et al., 2002; Karbowniczek et al., 2004, 2006), and so do PDK1 and GSK3 on MEK and ERK kinases, respectively (Sato et al., 2004; Wang et al., 2006). Finally, PTEN inhibits MAPK through the negative regulation of integrin signaling (Gu et al., 1998).

Importantly, PI3K regulates the activity of Rac G monomeric proteins through the modulation of Rac-GEFs by PIP3 (Welch et al., 2003). This cross-talk is of importance for the regulation of actin cytoskeleton and other functions such as cell cycle and transcription. Conversely, several studies point out to the regulation of p85 by Rac in vitro, which might be of importance for neutrophil chemotaxis (Welch et al., 2003).

PI3K pathway can also regulate JNK pathway. AKT phosphorylates and inhibits apoptosis signal-regulating kinase 1 (ASK1) at Ser83, thereby promoting cell survival (Yuan et al., 2003; Zhang et al., 2005; Wu et al., 2006). On the other hand, the group of Sawyers recently identified JNK pathway activation as a major consequence of PTEN loss in an elegant screening, suggesting that PI3K induces a parallel activation of AKT and JNK to promote cancer progression (Vivanco et al., 2007).

Overall, the multiple cross-talks arising from PI3K pathway toward other signaling cascades suggest that many of the PI3K-regulated functions actually go beyond PI3K canonical signaling.

Signaling pathways regulating PI3K pathway

PI3K pathway receives regulatory signals from other pathways at the transcriptional and post-translational level (Figure 3). PTEN transcription is regulated by RAS and JNK pathway in cancer cells leading to tumor progression (Beck and Carethers, 2007; Vasudevan et al., 2007; Xia et al., 2007). Overexpression of mutant Ras promotes a MAPK-dependent repression of PTEN transcription (Vasudevan et al., 2007). A MEK-dependent pathway increase c-Jun levels and increases the binding of this transcription factor to the promoter of PTEN (Hettinger et al., 2007; Vasudevan et al., 2007). The downregulation of PTEN by MAPK pathway promotes the escape from apoptosis of Ras-overexpressing cells. In another study, PTEN was found downregulated in a subset of lung cancer cell lines (Xia et al., 2007). This downregulation of PTEN correlated with the overexpression of the JNK regulator MKK4, which increased NF-κB activity and binding to PTEN promoter. Therefore, PTEN downregulation might be a mechanism of other pathways to escape apoptosis or promote proliferation. Indeed, in T-cell acute lymphoblastic leukemia, aberrant Notch pathway represses PTEN transcription through induction of HES-1 in turn providing a novel therapeutic approach for the treatment of this malignancy with the combined inhibition of PI3K and Notch pathways (Palomero et al., 2007).

Figure 3
figure3

Pathways that regulate PI3K signaling. PI3K pathway (green) is under the control of other cascades (yellow). Ras-MAPK pathway modulates this pathway at multiple levels (MAPK–c-Jun–PTEN, Ras–PI3K, ERK–TSC2, RSK–TSC2, RSK–S6, RSK-eIF4B) and so does MKK4–JNK pathway through the activation of NF-κB. Lastly, PML inhibits PI3K signaling at the level of AKT and mTORC1. PI3K, phosphoinositide 3-kinase; PML, promyelocytic leukemia.

With regards to PI3K regulation, class I and II PI3K isoforms have a RBD by which Ras facilitates PI3K anchorage to the membrane and thus its full activation (Engelman et al., 2006). Ras binding to PI3K is required to transmit EGF and FGF2 extracellular signals, which accounts for a primary role of Ras in the regulation of PI3K signal transduction (Ramjaun and Downward, 2007). Moreover, the oncogenic ability of Ras is linked to its ability to activate this pathway (Rodriguez-Viciana et al., 1997; Gupta et al., 2007). The expression of a mutant dominant-negative form of p85 (Rodriguez-Viciana et al., 1997) or the mutation of PI3K1A in the RBD (Gupta et al., 2007) reduces Ras-mediated transformation and the aggressiveness of lung tumors in Ras mutant transgenic mice. The use of these mutant mice provided as well interesting data about the tissues that might be more sensitive to the Ras–PI3K cross-talk. As an example, the observed abnormalities of PI3K mutant mice in the development of the lymphatic system suggest that PI3K requires to bind Ras to regulate this function, at least partially through the proper activation of VEGFR3, TIE2 and FGF2 receptors (Ramjaun and Downward, 2007).

The interaction between Ras and PI3K is also critical for correct cell polarity and migration. Studies carried out in Dictyostelium and hippocampal neurons show that the proper cross-talk of these two pathways is essential for axon formation and cell polarity and migration (Funamoto et al., 2002; Sasaki et al., 2004, 2007; Fivaz et al., 2008). Moreover, in Dictyostelium, cytoskeleton components such as F-actin filaments play an important role in the signaling between PI3K and Ras, promoting their correct localization and interaction in the plasma membrane (Sasaki et al., 2007). Therefore, the PI3K, at the level of PI3K and PTEN, plays a critical role in cell polarity, chemotaxis and migration, which requires a tightly regulated cross-talk with Ras pathway.

TSC complex is also regulated by MAPK at two different levels. On the one hand, p90RSK1 phosphorylates and inhibits TSC2 at Ser1798, thereby promoting mTORC1 activation (Roux et al., 2004). On the other hand, a study from our group has recently shown that ERK can also directly phosphorylate TSC2 on Ser664, leading to the inhibition of the complex (Ma et al., 2005a). Thus, the regulation of mTORC1 signaling by MAPK might be an important component of tumors with aberrant Ras activation to progress. The study of this cross-talk in cancer specimens reveals that tumors with hyperactive ERK often show TSC2 phosphorylation on Ser664 and concomitant activation of mTORC1, whereas in a second group of patients, mTORC1 is found overactive even in the absence of TSC2 phosphorylation on this site (Ma et al., 2007).

Lastly, PI3K and MAPK signals also converge downstream of mTORC1. p90RSK phosphorylates the ribosomal protein S6 at Ser235/236 therefore promoting its activation upon Ras–MAPK upregulation (Roux et al., 2007). Additionally, S6K and RSK phosphorylate eIF4B at Ser422, thus enhancing its interaction with the eIF3 and promoting protein translation (Shahbazian et al., 2006).

Therefore, a more detailed understanding of MAPK and PI3K cross-talk in cancer, and the subsequent stratification of patients depending on the input source of mTORC1 activation will allow individualized therapy geared to singly or combinatorially target specific pathways, thus enhancing the anticancer potency of these treatment modalities, as we discuss in the following paragraph.

Clinical implications

Targeting PTEN/PI3K pathway represents a major aim for the treatment of a number of diseases. Indeed, several compounds are now being designed and tested in preclinical trials for the treatment of cancer with hyperactive PI3K (Luo et al., 2003), such as the PI3K SF1126 from Semaphore and XL147 from Exelixis. Although the first generation of PI3K inhibitors derived from wortmannin and LY294002 displayed high toxicity, second generation wortmannin derivatives may prove much more effective (Harvey and Lonial, 2007; Garlich et al., 2008; Yuan and Cantley, 2008).

Tremendous effort has also been invested at targeting molecules operating downstream of the PTEN/PI3K pathway. mTORC1 has been probably the most studied target of the PI3K pathway, perhaps due to the original identification of rapamycin (Guertin and Sabatini, 2007). But, the efficiency of this family of compounds in clinical trials has been less than satisfying. mTORC1 inhibitors failed to inhibit tumor progression in a variety of cancers, with the exception of kidney cancer, TSC-associated symptoms (Faivre et al., 2006) and brain tumors (Cloughesy et al., 2008). With the discovery of mTORC1 negative feedbacks, it became evident that the use of mTORC1 inhibitors as single agents might be limited, whereas the combinatorial use of inhibitors of other pathways would be required to target a particular feedback in stratified patients (Figure 4).

Figure 4
figure4

Therapeutic manipulation of PTEN/PI3K feedbacks and cross-talks in the treatment of human disease. PI3K, mTORC1 and MAPK conform a signaling triangle composed of feedbacks and cross-talks. Therefore, the use of selective inhibitors for a given pathway may have dramatic consequences on the other members of this triangle. This relationship highlights the requirement of patient stratification and the use of dirty drugs or drug combination for the increase in effectiveness in the treatment of a particular disease. PI3K, phosphoinositide 3-kinase.

In principle, drug combinations for the inhibition of main survival pathways (mTORC1, PI3K and MAPK) might result in high toxicity due to the targeting of critical functions in non-transformed cells. However, tumor cells appear to be surprisingly more sensitive to these treatments. These data have been rationalized putting forward the hypothesis that tumor cells are addicted to oncogenic events (Weinstein and Joe, 2008). The specific genetic hit driving transformation (that is, PTEN loss) in a cell would promote the re-adjustment of the intracellular wiring (MAPK, translation, response to growth factors) to the aberrant activation of the pathway (PI3K), therefore resulting in a higher dependency on this cascade for cell homeostasis. The use of pharmacological inhibitors would in turn be more effective in the cancer cell as this perturbation would result in a further disequilibrium to the overall cell signaling network. This hypothesis provides support for the use of drug combinations that would inhibit a target pathway preventing feedback or cross-talks.

The concept of signaling cross-talks and drug combinations has promoted the design of the so-called ‘dirty drugs,’ which can target multiple molecules or multiple kinases at once. To this end, the design of compound mTOR kinase-PI3K inhibitors would be extremely useful in targeting in full both upstream and downstream of the PI3K pathway in the absence of undesirable feedback regulations, as modeled by recently developed compounds by Novartis (NVP-BEZ235) and Exelixis (XL765) (Garcia-Echeverria and Sellers, 2008). In this respect, it has been proposed that the activity of rapamycin on mTOR may go beyond mTORC1 at higher dose, where AKT activation through the disassembly of mTORC2 would be prevented as well (Guertin and Sabatini, 2007). Although this possibility needs to be further explored, it is tempting to speculate that mTOR kinase inhibitors could provide a double edge sword toward inhibition of the PTEN/PI3K pathway from top (mTORC2) to bottom (mTORC1).

Concluding remarks

Since the initial discovery of the PI3K pathway, multiple new components have entered this network, thus increasing its complexity. Although represented as a linear/up-to-down cascade, in the past few years the numerous feedback regulations have put the pathway literally upside down. From PTEN to mTOR, each member of this network is either feedback-regulated or cross-talks to other signaling cascades. Therefore, the characterization of the context and genetic background where a particular signaling circuit is operational is essential both for the understanding of cell physiology and also for the appropriate design of therapeutic intervention in pathological conditions.

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Acknowledgements

We thank all members of the Pandolfi lab who contributed to these ideas. In particular, we are grateful to L Salmena and MS Song for insightful discussions. The work of PPP was supported by the US National Cancer Institute, and AC was supported by a European Molecular Biology Organization Long-Term Fellowship Award.

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Correspondence to P P Pandolfi.

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The authors declare no competing financial interests.

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Carracedo, A., Pandolfi, P. The PTEN–PI3K pathway: of feedbacks and cross-talks. Oncogene 27, 5527–5541 (2008). https://doi.org/10.1038/onc.2008.247

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Keywords

  • feedback
  • PTEN
  • PI3K
  • drug combination
  • cross-talk

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