CHAPTER 1
The PI3K/Akt/PDK1 pathway
1.1. The PI3K/Akt/PDK1 pathway
1.1.1 Physiological role
The PI3K/Akt/PDK1 pathway is an important and intricate signalling network that regulates several cellular mechanisms, in which many proteins have different and notably role. This complexity highlights the importance of each nodes involved in the pathway.
As a consequence of the stimulation with growth factors like Epidermal growth factor (EGF) or insulin-‐like growth factors (IGFs) and cytokines on receptor tyrosine kinase (RTKs), Akt is recruited from cytosol to plasma membrane and phosphorylated at two key regulatory sites, Thr308 and Ser473 by PDK1 and mTORC2, respectively. Akt activation depends on phosphatidylinositol 3,4,5-‐trisphosphate (PIP3) levels, a
second messenger produced by phosphoinositide 3-‐kinase (PI3K) through phosphorylation on 3ʹ′-‐position of the inositol ring in phosphatidylinositol 4,5-‐bisphosphate (PIP2). The interaction of PIP3
with the pleckstrin homology (PH) domain of Akt promotes the translocation of Akt to the plasma membrane, where it undergoes
phosphorylation by PDK1. This binding also induces a conformational shift, which transform the Akt “close” form to the “open” one, thus allowing for PDK1-‐mediated phosphorylation of Akt Thr308. This phosphorylation is sufficient to activate some Akt downstream substrates, but additional phosphorylation in the C-‐terminal domain at serine Ser473 by mTORC2 is required for the full activation of Akt [1]. The activity of PI3K is countered by phosphatase and tensin homolog protein (PTEN), a tumor suppressor gene that converts back PIP2 in PIP3
thus switching off the activated pathway (Figure 1.1).
The activated Akt stimulates cell growth and survival by phosphorylating numerous downstream substrates responsible for the final biological response.
Inactivation of proteins like caspase-‐9 and BCL antagonist of cell death (BAD), two apoptosis activating factors, is linked to the cell survival through apoptosis inhibition. The pathway also regulates cellular energy control and glucose metabolism through glycogen synthase kinase-‐3 (GSK3) and phosphofructokinase 2 (PFK2), meanwhile the interaction with Forkhead box transcription factors (FoxO), p21 and p27 lead to proliferation effect.
Akt is also double-‐strand linked to the mammalian target of rapamycin (mTOR) through a positive feedback mechanism, another important kinase involved in growth and cell proliferation [2].
The PI3K signalling regulates senescence and angiogenesis, and both processes are influenced by the activity of vascular endothelial growth factor (VEGF) and hypoxia-‐inducible factor (HIF-‐1α) [3].
Moreover phosphorylated Akt interacts with Mouse double minute 2-‐ homolog (MDM2), an important negative regulator of the p53 tumor suppressor.
Figure 1.1: Schematic representation of PI3K/Akt/PDK1 pathway: black arrows
indicate upstream Akt activators, while green and red arrows indicate, respectively, activated and inhibited Akt downstream effectors (modified from
https://www.caymanchem.com/app/template/Article.vm/article/2132).
1.1.2 Oncogenic role
Thus PI3K/Akt/PDK1 pathway plays a significant role in the cell: its activation triggers a chain phosphorylation that leads to a biological response. In physiological condition, this process regulates the normal cellular working, but, as a result of mutation, could lead to neoplastic cellular transformation. Indeed this pathway is frequently found inappropriately activated in various cancers and it is not surprising considering that its major cellular effects are mandatory hallmarks of the cancer.
Different genetic abnormalities were observed in this pathway in human cancer, including activating and deactivating mutations, copy number changes and post-‐transcriptional epigenetic irregularities [4]. Nevertheless the two major mechanisms of PI3K/Akt/PDK1 pathway activation are somatic alteration in specific nodes of the pathway, like PI3K and Akt, and activation by RTKs [3]. The role of these mutations in different cancer settings will be discussed within this Chapter.
Several studies have also demonstrated a connection between altered PI3K/Akt/PDK1 signalling network and multi drug resistance (MDR), the major problem in cancer treatment. Most of the more resistant cancer types show hyperactivation of this pathway.
In breast adenocarcinoma Knuefermann showed that the cell lines expressing both HER2 and HER3 had a higher phosphorylation level of Akt and were associated with increased MDR. Since selective inhibition of PI3K or Akt kinases activity leads to induction of apoptosis, the proposed mechanism for this synergy was the potentiation of apoptosis [5].
Emerging role of the KRAS-‐PDK1 axis was identified in pancreatic cancer, an highly aggressive tumour very resistant to treatments, generally diagnosed late because of absence of specific symptoms and strongly metastatic [6].
Also Glioblastoma Multiforme (GBM), known as the most common, most aggressive and resistant malignant primary brain tumor in humans [7], shows an increased activation of PI3K/Akt/PDK1 pathway. Correlation between this pathway and GBM will be discussed in detail in Chapter 2.
The importance of PI3K/Akt/PDK1 signalling network is demonstrated also by the remarkable results reached in clinical treatments with different molecules inhibiting one or more crucial components in this pathway. Indeed, the inhibition of such PI3K/Akt/PDK1 pathway components can restore sensibility to chemotherapy, radiotherapy, and hormonal treatment [3].
1.1.3 Raf/Ras/MEK/MAPK and PI3K/Akt/PDK1 signalling network
The Ras/Raf/MEK/MAPK pathway is a signalling cascade involved in numerous cellular functions such as: cell cycle regulation, wound healing and tissue repair, integrin signalling, cell migration but also angiogenesis. This pathway also converges with the PI3K/Akt/PDK1 pathway as they are both activated by Ras superfamily and they share numerous downstream effectors. Network constituted by the
5 connection of the pathways is showed in Figure 1.2. Since these two signaling pathways have been shown to play crucial roles in the transmission of proliferative signals from membrane bound receptors, they were investigated also for their responsibility in tumorigenesis. In some cases, resistance to therapy may be due to the activation of an additional pathway, able to balance the signal. Although the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways have distinct effects on cell proliferation, they have many common downstream targets that may promote survival in the absence of the corresponding functional pathway [8].
Figure 1.2: Interactions between the Ras/Raf/MEK/ERK and
Ras/PI3K/PTEN/mTOR signalling networks: green arrows indicate activation while red lines indicate inhibition activity (modified from [8]).
It was also demonstrated that the crosstalk between the Raf/Ras/MEK/MAPK and PI3K/Akt/PDK1 pathways is involved in the maintenance of self-‐renewal and tumorigenicity of GBM stem-‐like cells [9].
Figure 3. Interactions between the Ras/Raf/MEK/ERK, Ras/PI3K/PTEN/mTOR and Wnt/ Catenin Pathways that Result in the Regulation of Protein Translation and Gene Transcription. The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR
pathways can affect protein translation by complex interactions regulating the mTORC1 (grouped together in a purple box) and mTORC2 (grouped together in a blue box) complexes. GF stimulation results in GFR activation which can activate both the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways. Akt can phosphorylate and inhibit the effects of GSK 3 , TSC2 and PRAS 40 (indicated in red ovals), which result in mTORC1 activation. ERK and PDK1 can phosphorylate p90Rsk1
(indicated in green ovals), which in turn can phosphorylate and inhibit TSC2 (indicated in red oval). Akt mediated phosphorylation of GSK 3 also affects the Wnt/ catenin pathway and EMT. Rapamycin targets mTORC1 and inhibits its activity and also results in inhibition of downstream p70S6K. The effects of rapamycin are complex as long term administration of rapamycin may prevent mTOR from associating with mTORC2 and hence full activation of Akt is prevented. However, rapamycin treatment may result in activation of PI3K, by inhibiting the effects of p70S6K on IRS 1 phosphorylation which results in PI3K and Akt activation. Also rapamycin treatment may result in the activation of ERK in some cells, presumably by inhibition of the p70S6K mediated inhibition of IRS1. These later two effects of rapamycin could have positive effects on cell growth. Energy deprivation will result in the activation of serine/threonine kinase 11 (STK11 a.k.a LKB1) and AMP kinase (AMPK) which can result in TSC2 activation (indicated in red ovals) and subsequent suppression of mTORC1. In contrast Akt can phosphorylate and inhibit the activity of AMPK. Inhibition of PDK 1 activity can also result in activation of mTORC1, presumably by suppression of p70S6K and hence inhibition of IRS1 (indicated in red oval) effects on PI3K activity. The PTEN, TSC1, TSC2 and LKB1 tumor suppressor genes all converge on the mTORC1 complex to regulate protein translation. Thus the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways can finely tune protein translation and cell growth by regulating mTORC1. Rapamycin can have diverse effects on these processes. Also these pathways can interact with the Wnt/ catenin pathway which is important in developmental processes, EMT and CICs. Upon activation of the Wnt pathway, catenin forms a complex with Bcl 9, PYGO, plakoglobulin and TCF/LEF which result in the transcription of critical genes including cyclin D1, c Myc, SALL4 and PPAR .
1.2. PI3K
The class I phosphoinositide 3-‐kinases (PI3Ks) are lipid kinases that regulate a broad range of essential functions including growth, proliferation, and migration.
PI3Ks specifically catalyze the phosphorylation on 3-‐position of the inositol ring in specific phosphoinositides (PtIns). PtIns are phospholipids formed by two fatty acid chains and a water-‐soluble inositol head-‐group linked by a glycerol backbone. The lipid products of PI3Ks can act as cellular second messengers since they activate several proteins regulating their intracellular localization or their conformational changes [10].
1.2.1 Structure
Mammalian PI3K exists in eight isoforms and they are grouped into three classes based on their structural and biochemical features:
All PI3K isoforms possess a catalytic subunit, the PI3K core, consisting of a C2 domain, a helical domain and a catalytic domain (Figure 1.3).
Class I PI3K can be further divided into two subclasses: IA and IB. They are dimers of one catalytic and one regulatory subunit; moreover all class I catalytic subunits possess a Ras-‐binding domain.
Class IA, which are mainly activated by RTKs, comprises distinct catalytic subunits (p110α, p110β, p110δ and p110γ); the class IA Ras-‐binding domain subunits also specifically possess a p85-‐binding domain that mediates interactions with the regulatory subunits (for p110α, p110β and p110δ) or p101 or p87 (for p110γ). All p85 isoforms have two Src homology 2 (SH2) domains [10, 11].
Class IB PI3Ks are mainly activated by GPCRs (G-‐protein-‐coupled receptors); this subfamily contains the p110γ as the only catalytic subunit.
Class II PI3Ks is a monomer of high molecular weight, which can exist in mammals in three isoforms: PI3K-‐C2α, PI3K-‐C2β and PI3K-‐C2γ. Class II PI3Ks lack the regulatory-‐subunit-‐binding domain, while they possess a Ras-‐binding domain and the PI3K core (Figure 1.3). Moreover, they
contain a C-‐terminus extension, constituted by a Phox homology domain, and a second C2 domain. The N-‐terminus extensions are distinct within the class II isoforms (clathrin-‐binding region in PI3K-‐C2α and proline-‐rich sequences in PI3K-‐C2β), suggesting specific roles for this region [10].
Class III PI3K has one catalytic member, Vps34 (vacuolar protein sorting 34). This class is a monomer that lacks the Ras-‐binding and the regulatory-‐subunit-‐binding domains and catalyses specifically the synthesis of Phosphatidylinositol 3-‐phosphate (PI3P) [9].
Figure 1.3: PI3K isoforms structures (modified by [11]).
1.2.2 Physiological role
PI3Ks can be activated by multiple signalling pathways, including tyrosine phosphorylated receptors and their adaptors, GPCRs, and the Ras superfamily. Each PI3K isoform has a different role based on their ability to integrate signals from these inputs: among them, class I are the widest featured one.
Class I PI3Ks play remarkable roles in regulating cellular processes and, not surprisingly, they have been frequently found misregulated in human diseases. Class I PI3Ks catalyse the phosphorylation of PIP2, the
primary substrate, at 3-‐position of the inositole ring, converting it into PIP3 (Figure 1.4).
8
PIP3, the main in vivo lipid product of class I PI3Ks, is the most studied
PI3K effector and a very well established second messenger, involved in many cellular functions. PIP3 is able to recruit PH-‐domain-‐containing
proteins to the inner surface of the cell membrane. PH domains is a lipid-‐binding modules contained in a range of proteins, including several cytoplasmic kinases such as Akt [12].
The mechanism described is counteracted by tumor suppressor phosphatase and tensin homolog PTEN, a phosphatase that dephosphorylate back PIP3 to PIP2.
Figure 1.4: Schematic representation of PI3K structure and function (modified
from [12]).
1.2.3 PI3K in cancer
Since its discovery 20 years ago, several studies have established the central role of PI3K/Akt/PDK1 in cancer growth, metabolism, survival, and motility. The hyperactivation of this pathway seems to affect 30– 50% different types of human cancers [13].
Oncogenic activation of the PI3K/Akt/mTOR pathway can occur in multiple ways, such as point mutations of PI3K, inactivation of PTEN, and amplification or mutation of Akt. Particularly, in 2004, point mutations in PIK3CA, the gene encoding the p110α subunit of PI3K, were discovered among the most commonly documented mutations in cancer. These modifications are able to enhance PI3K activity. Consequently, the expression of p110α mutants in cells induces the Akt
R E V I E W S
human cancers (see below). Once it became clear that PTEN functions primarily as a PIP3 lipid
phos-phatase12–16, the central importance of PIP
3regulation in
cancer became indisputable. Although PTEN might also have activity against protein substrates17,18, the
evi-dence from mutational studies and from analysis of the hereditary cancer syndrome COWDEN’S DISEASEindicates that the PIP3phosphatase activity is responsible for the tumour-suppressor function of PTEN(BOX 1).
The SHIP phosphatases also act on PIP3, but
remove phosphate from the 5-position rather than the 3-position, creating PtdIns(3,4)P2 (note that PtdIns(3,4)P2can also be generated by class II PI3Ks). PtdIns(3,4)P2can function as a second messenger (like PIP3) to recruit pleckstrin-homology (PH)-domain -containing proteins, such as AKT (see below). So, although both PTEN and SHIP reduce the level of PIP3
in cells, PTEN seems to have primary responsibility for controlling the mitogenic effects of phosphoinositides because it reduces the levels of all those phosphorylated at the D3 position. As expected, knockout mutations in Pten, but not Ship1, give a strong cancer phenotype in mice. Although useful, this model is likely to be an oversimplification. PIP2— the product of the PTEN
reaction — might be a second messenger in its own right, as well as being a substrate for several other phos-phinositides that have signalling functions. In addition, Ship1-knockout mice can develop myeloproliferative syndromes, indicating that PtdIns(3,4)P2can activate
certain mitogenic pathways19–21.
Downstream of PIP3: the AKT pathway
Now that the central role of PIP3in cancer seems clear, there is renewed emphasis on defining precisely how PIP3functions as a second messenger. Much of
the recent progress is based on the concept that PIP explain much of the current experimental data, but
there are many other potential modes of regulation. Precisely how the various isoforms and splice variants of p85 and p110 affect PI3K activity remains to be determined. In addition, we have a limited under-standing of how the activated PI3K complex is downregulated. One hypothesis is that tyrosine phos-phorylation of p85, which occurs after the p85–p110 complex has been recruited to the active RTK, serves as a negative regulatory signal that leads to a reduction in p110 catalytic activity9. A better understanding of
these details will undoubtedly provide new insights and opportunities for pharmacological intervention in PI3K-pathway-driven cancers.
PIP3phosphatases
The primary consequence of PI3K activation is the gen-eration of PIP3in the membrane, which functions as a second messenger to activate downstream pathways that involve AKT and other proteins, as described below. PIP3levels are barely detectable in mammalian cells under unstimulated growth conditions and are tightly controlled, owing to the combined effects of stringent PI3K regulation and the action of several PIP3
phos-phatases (PTEN,SHIP1and SHIP2) (FIG. 1). The PIP3
phosphatase that is most clearly involved in oncogenesis is PTEN (also called MMAC1), a 3-position lipid phos-phatase that converts PIP3back to PIP2. This control mechanism is analogous to the regulation of GDP- ver-sus GTP-bound RAS through the opposing effects of guanine nucleotide exchange factors (GEFs; the activators) and GTPase-activating proteins (GAPs; the repressors). PTEN was isolated originally as a tumour-suppressor gene in breast cancer and glioblastomas using traditional positional-cloning strategies10,11, and has
subsequently been implicated more broadly in various COWDEN’S DISEASE
A hereditary predisposition to tumours — especially hamartomas of the skin, mucous membranes, breast and thyroid — that is caused by PTEN mutations.
Figure 1 |Minding your Ps: the PtdIns(4,5)P2–PtdIns(3,4,5)P3cycle. Phosphatidylinositol phosphates are composed of a
membrane-associated phosphatidic acid group and a glycerol moiety that is linked to a cytosolic phosphorylated inositol head group. Phosphatidylinositol 3-kinase (PI3K) can phosphorylate PtdIns(4,5)P2(PIP2) at the D3 position to form the second messenger
PtdIns(3,4,5)P3(PIP3). Phosphorylation at the D3 position is necessary for binding to the pleckstrin-homology domain of AKT (not
shown). Dephosphorylation of PIP3to regenerate PIP2is accomplished by the 3-phosphatase PTEN. Additionally, PIP3can be dephosphorylated at the D5 position by SHIP1 or SHIP2 to generate PtdIns(3,4)P2, another potential second messenger.
O O O O P P P P P P P P P HO HO 1 6 OH 5 4 3 2 O O O O HO OH 1 6 5 4 3 2 Fatty acids Glycerol backbone Inositol head group P P P O O O O HO OH OH 1 6 5 4 3 2 p110 (α,β,δ) p85 (α,β)
PIP2 PI3K PIP3 PtdIns(3,4)P2
ATP ADP
PTEN
SHIP1/2
activation in the absence of growth factor stimulation, thus leading to tumor development. Although these mutations have been described in almost every major tumor types, they were mainly found in breast, colon, and endometrial cancers and glioblastoma [3, 14].
Moreover, since PTEN is able to counterbalance the PI3K activity, it is not surprising that mutations of this tumor suppressor gene represent the second most common genetic abnormalities found in human cancer. For instance, the majority of pediatric gliomas show activation of the PI3K/Akt/PDK1 pathway [3].
Despite recurrent mutations in PI3Kα, PI3Kδ and PI3Kγ abnormal form are uncommon. PI3Kδ is preferentially expressed in leukocytes and it is important in B cell activation, proliferation, survival, and lymphoid tissue homing. PI3Kδ signalling was demonstrated to be particularly hyperactive in many B cell malignancies. Several preclinical studies showed that isoform-‐specific inhibition of PI3Kδ resulted in cytotoxicity of B cells while sparing toxicity to other hematopoietic cell types. Also PI3Kγ is preferentially expressed in leukocytes and is central to the growth and survival of B cell malignancies. PI3Kγ is also known to have a role in the maintenance of the tumor microenvironment and involved in T cells development and survival [13].
1.2.4 PI3K inhibitors
Since PI3K exists in different isoforms, inhibitors are classified according to their selectivity: pan-‐PI3K inhibitors, able to inhibit different isoforms with various potency, and PI3K isoform specific-‐inhibitors.
Pan-‐PI3K inhibitors target all Class IA PI3Ks and are represented by
several small molecule drugs XL147, PX-‐866, BKM120, GDC-‐0941, BAY806946, GSK2126458, and CH5132799, and one RNA interfering (RNAi) agent, ATU027.
XL147, a selective inhibitor of Class I PI3K isoforms, is now in phase II
clinical trial, both in monotherapy and in combination. PX-‐866 is a derivative of Wortmannin in phase II clinical trial and acts as an irreversible pan-‐PI3K Class IA and B inhibitors. BAY806946 (BAY) is a potent and highly selective reversible pan-‐Class I PI3k inhibitor in phase I trial.
BKM120 is the most advanced PI3K inhibitor in clinical trial (phase 4). It
is a highly specific pan-‐Class I PI3K inhibitor and it does not affect other proteins such as mTOR or Vps34. It showed an IC50= 52, 166, 116, 262
nM versus p110 α, β, γ, δ respectively.
GDC-‐0941 is another pan-‐Class I PI3K inhibitor belonging to an early
generation PI3K inhibitor, actually in Phase II. It showed IC50 values
lower than BKM120 (IC50= 3, 3, 33, 75 nM versus p110 α, β, γ, δ
respectively).
Finally ATU027 is a novel RNAi therapeutic that targets N3 (PKN3), a protein kinase C-‐related, acting as downstream effector of the PI3K signaling pathway. Currently it is subjected to a phase I trial in cancer patients and, if successful, ATU027 and other related siRNA-‐based therapeutics could be a novel approach for targeting individual components of the PI3K pathway.
The PI3K isoform-‐specific inhibitors selectively inhibit PI3K p110 α, β, γ or δ catalytic subunits; this feature may be an advantage since this molecules could inhibit more completely the target with fewer side effects.
CAL101 is a specific inhibitor of PI3Kδ, as mentioned before
predominantly expressed in leukocytes, with a potency of 2.5nM. Early trials generated impressive response rates in some lymphomas. The compound is generally well tolerated. Actually phase II/III trials in B-‐cell malignancies are ongoing [3].
Figure 1.5: PI3K inhibitors in different phases of clinical trial.
1.3. The Akt/PKB kinase
The Protein Kinase B or Akt belongs to the AGC kinase family, the most evolutionary conserved groups among human protein kinases. The AGC kinase group was named after three representative families, the cAMP-‐ dependent protein kinase (PKA), the cGMP-‐dependent protein kinase (PKG) and the protein kinase C (PKC) families [14].
1.3.1 Structure
Akt, also known as protein kinase B (PKB), was originally identified as the cellular homologue of the viral oncogene, v-‐akt [15]: a serine/threonine kinase similar to protein kinases A and C. Akt acts as a
H N O NH2 S O O NH N N NH Cl O XL#147 O O Me H AcO Me O OH N O H3CO PX#866 N H2N F F N N N N O O BKM120 N N N S O N N SO O GDC0941 O N O N N N N H O N N NH2 O BAY806946 S O O N N N N F F O GSK2126458 N N H2N N N N O N S O O CH5132799 N N O F HN N N N NH CAL101
central core that transduces various extracellular signals to regulate a wide range of biological processes through phosphorylation of distinct protein substrates.
Mammalian Akt exists as three different isoforms, Akt1, Akt2, Akt3, which share high sequence and structural homology. Their structure consists of an amino-‐terminal pleckstrin homology (PH) domain and a carboxyl-‐terminal regulatory domain (CTD). Belonging to class of Ser-‐Thr kinases, Akt isoforms present two different phosphorylation site both necessary to have a complete activation: Thr is situated in the Kinase domain (308 in Akt1, 309 in Akt2, 305 in Akt3) while Ser is positioned in the CTD (473 in Akt1, 474 in Akt2, 472 in Akt3) (Figure 1.6).
Figure 1.6: Akt isoforms.
However accumulating evidence suggests differential substrate specificity and biological functions among Akt isoforms: Akt1 is ubiquitously expressed in various tissues at high levels; Akt2 is highly expressed in the muscle, liver and adipose tissues while modestly expressed in other tissues; Akt3 is predominantly expressed in brain and testis. Since these Akt isoforms are activated by PI3K and PDK1 in a similar way and share also some downstream effectors, it is proposed that Akt isoforms functions may be partially overlapped. Therefore, these observations lead researchers’ attention to investigate more deeply the roles of each Akt isoform in physiopathological conditions [1].
Numerous studies have established that growth factors (such as EGF, Heregulin and insulin) hormones and cytokines stimulate Akt activation
through serial phosphorylation events. The binding of ligand to its associated RTK causes PI3K activation that converts phospholipid PIP2 to
PIP3. Then PIP3 interacts with the PH domain and recruits both Akt and
PDK1 to the plasma membrane, where PDK1 can phosphorylate Akt Thr308 in the activation loop, consequently changing Akt in its active conformation. While this phosphorylation is enough to activate some Akt downstream substrates, a second phosphorylation by mTORC2 at Ser473 in the C-‐terminal domain completely activates Akt. Phosphorylation at Akt Ser473 seems to be modulated also by other kinases such as DNA-‐dependent protein kinase (DNA-‐PK), integrin-‐linked kinase (ILK), and mitogen-‐activated protein kinase-‐activated protein kinase-‐2 (MAPKAPK2) in various contexts [1].
Conversely, as previously discussed, PTEN dephosphorylates PIP3 thus
preventing Akt membrane recruitment and phosphorylation. As well as PTEN, also other two phosphatases, protein phosphatase 2A (PP2A) and PH domain and leucine rich repeat protein phosphatase (PHLPP), counteract Akt activation throughout the dephosphorylation of Akt at Thr308 and Ser473, respectively.
1.3.2 Physiological role
Akt regulates critical cellular processes activating or inhibiting a variety of downstream targets. Each target often fulfills different cellular functions including cell survival and proliferation, glucose metabolism, cell migration, cancer progression and metastasis.
For instance, FoxOs, once phosphorylated by Akt, are sequestered in the cytoplasm where they fail to induce transcription of genes associated with apoptosis and cell cycle arrest, thus blocking the apoptosis mechanism. Akt phosphorylation of GSK3β induced cells to reacquire survival features, since this kinase is known to downregulate protein substrates for cell survival and proliferation. The phosphorylation of the Tuberous Sclerosis Complex 2 (TSC2) allows the reactivation of mTORC1, a protein complex essential for protein translation and cell growth normally blocked by TSC2. Akt phosphorylation of Bad, a pro-‐apoptotic
protein that controls mitochondrial outer membrane permeability, decreases Cytochrome C release to protect cells from apoptosis.
Moreover, Akt activates some substrates such IKKα, Skp2 and AS160. The phosphorylation of IKKα leads to the induction of immune response through upregulation of the necrosis factor-‐κB (NF-‐κB). The activation of Skp2, a protein essential for cell cycle progression, migration and metastasis, facilitates its cytosolic translocation, thus stimulating cell migration and cancer metastasis [1]. Again, the AS160 activation (Akt substrate, 160 kDa), promotes Glut4 translocation and glucose uptake in muscle cells.
Additionally, Akt phosphorylates MDM2 promoting its translocation to the nucleus, where it negatively regulates p53-‐induced apoptosis. Akt mediated-‐phosphorylation decreases the protease activity of caspase-‐9, activated during programmed cell death, but also inhibits p27 expression preventing its cell-‐cycle inhibitory effects.
Finally, Akt activates endothelial nitric oxide (NO) synthase (eNOS) through its direct phosphorylation. The release of NO produced by activated eNOS can stimulate vasodilation, vascular remodeling, and angiogenesis [16].
A summary panel of all Akt substrates and relative cellular role is proposed in Figure 1.7.
Figure 1.7: Main Akt direct substrates and relative roles (modified from [16]).
1.3.3 Akt in cancer
Since AKT/PKB is involved in numerous cellular processes, reasonably its deregulation leads to different disorders like cancer, diabetes, cardiovascular and neurological diseases.
Although mutations in Akt are rarely found, Akt signaling is one of the most frequently hyperactivated pathways in many human cancers. Several mechanisms accounting for its aberrant activation have been proposed. These include mutation or amplification of PIK3C gene that encodes PI3K, loss of PTEN function and activation of RTKs.
The PI3K-‐dependent Akt serine/threonine phosphorylation by PDK1 and mTORC2 has long been thought to be the primary mechanism responsible for Akt activation. However, this regulation alone does not sufficiently explain how Akt hyperactivation can occur in tumors with normal levels of PI3K/PTEN activity. Mounting evidence demonstrates that aberrant Akt activation can be attributed to other posttranslational modifications, which include tyrosine phosphorylation, O-‐GlcNAcylation (glicosilation), as well as lysine modifications. Thus, the identification of proteins accountable for these modifications will provide important insights for cancer treatment [1].
Even though Akt isoforms partially share their functions, there are rising evidences that distinctive isoforms have non-‐overlapping effects in cancer. The single amino acid substitution E17K in the Akt1 PH domain has been identified in various human cancers such as breast, colorectal, endometrial, ovarian cancers and in some melanomas. Moreover Akt2 overexpression has been observed in colorectal cancers and metastases. Mutations in various Akt isoforms suggested a potential role for Akt inhibitors in therapy [3].
1.3.4 Akt Inhibitors
There are two main classes of Akt inhibitors in clinical development: PH-‐
domain, ATP-‐competitive and allosteric Akt inhibitors.
Perifosine is a PH domain inhibitor that targets Akt activity via
IC50=4.7µM and it has been proven as the most clinically advanced Akt
inhibitor. However, perifosine, such as other PH domain Akt inhibitors, lacks of selectivity since it inhibits not only Akt, but also PH-‐domains of other proteins. Consequently, the limited specificity of PH domain inhibitors as well as the potential for severe side effects (such as hemolysis) represents an important restrictive factor for the clinical use of this class of drugs. [1]. Nevertheless, perifosine has been evaluated in multiple phase I/II clinical trials both alone and in combination with various other agents; actually it is in phase III clinical trial. The most common adverse reactions are fatigue and gastrointestinal toxicity [3].
Triciribine phosphate (TCN-‐P) is a potent pan-‐AKT inhibitor
(IC50=130nM) in phase I/II clinical trials. It was shown to interact with
the PH domain of Akt and to interfere with its localization to the membrane, thereby preventing Akt phosphorylation and its subsequent activation [17].
TCN-‐P was administered to subjects whose tumors displayed evidence of increased Akt phosphorylation (p-‐Akt), as measured by immunohistochemical analysis. Modest decreases in tumor p-‐Akt were detected following treatment with TCN-‐P. No Maximum Tolerated Dose was determined, and no objective response was observed [3].
Pan-‐Akt kinase inhibitors are ATP-‐competitive Akt inhibitors. Because of
the highly conserved ATP-‐binding pocket within different kinases, this class of Akt inhibitors also affects other AGC kinases such as p70S6 kinase, protein kinase C (PKC) and Rho kinase. It is still to establish whether such an inhibitory profile will result in undesirable side effects in clinical settings.
Among them there is AZD5363, a potent pan-‐Akt kinase inhibitor possessing pharmacological properties reliable with Akt inhibition (IC50
Akt1=3nM, Akt2=8nM, Akt3=8nM). It inhibited the growth of a range of human tumor xenografts and caused significant regression in combination with docetaxel, particularly in breast cancer xenografts. AZD5363 is currently being investigated in phase I clinical trials.
Allosteric Akt inhibitors bind to the PH domain of the Akt enzyme,
forming a complex able to block the conformational change essential to Akt phosphorylation. Thus the translocation of Akt to the plasma membrane, essential for Akt activation, is disrupted. This mechanism of action might help to overcome the issue of kinase selectivity often observed with classic ATP-‐competitive inhibitors.
MK-‐2206 is an allosteric Akt inhibitor able to selectively inhibit Akt1, 2,
and 3 isoforms in nanomolar concentration (IC50: Akt1=8nM,
Akt2=12nM, Akt3=65nM). Changes in p-‐Akt before and after treatment was measured in tumor biopsies as well as surrogate tissues, evidencing inhibition of the target. MK-‐2206 is in phase II clinical trials.
Figure 1.8: Akt inhibitors in different phases of clinical trial.
Among the other Akt inhibitors not covered in the previous groups, there are PBI-‐05204 and RX-‐0201.
PBI-‐05204 (phase I/II clinical trial) contains oleandrin, a cardiac glycoside
that inhibits the α-‐3 subunit Na/K ATPase pump. Oleandrin inhibits the phosphorylation of Akt, but also the activation of NF-‐κB and export of fibroblast growth factor-‐2 (FGF-‐2). This compound also decreases mTOR activity through inhibition of p70S6K. Western blotting of peripheral blood mononuclear cells (PBMCs) showed that PBI-‐05204 markedly
HN HO Cl O N NH2 N N NH AZD5363 NH2 N N HN N O MK2206 N N N N N O HO OH NH2 O POHOH O TCN.P N+ O PO O -O Perifosine
The PI3K/Akt/PDK1 pathway
reduced phosphorylated Akt, p70S6K, and S6 levels in a time-‐dependent manner, suggesting Akt involvement.
RX-‐0201 (Archexin) is a 20-‐mer oligonucleotide with sequence
complementary to Akt-‐1 mRNA in phase I clinical trial.
1.4. PDK1, the master kinase of AGC
1.4.1 Structure
PDK1 kinase is a protein of 63kDa, constituted by 556 amino acids forming different domains: the N-‐terminal catalytic or kinase domain (71-‐359), the C-‐terminal Pleckstrin homology domain or PH domain (459-‐550), the hydrophobic domain, located in a region adjacent to the catalytic domain, and a nuclear export sequence. The nuclear export sequence is a four amino acids sequence, essential for transporting PDK1 from the cell nucleus to the cytoplasm through a nuclear transport mechanism that use a nuclear pore complex (Figure 1.9).
Figure 1.9: Schematic representation of PDK1 structure (modified from [4]).
The amino-‐terminal small lobe and the carboxy-‐terminal large lobe “sandwich” one adenosine triphosphate molecule essential for the subsequent substrate phosphorylation (Figure 1.x) [18]. The Glycine-‐rich loop, the αC-‐helix and an activation loop around the ATP-‐binding site of the enzyme are essential for the activity. These three regions are flexible and in motion in the inactive form, while they rearrange, organizing the ATP-‐binding site in its active form, able to catalyse phosphate-‐transfer reactions [19].
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sequence (Figure 1). The nuclear export sequence is a short sequence of four amino acids that are essential for exporting PDK1 from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport.
Similar to other AGC kinases, PDK1 possesses a phos-phorylation site within the activation loop (S241), which is phosphorylated in resting cells and is not affected by growth factor stimulation. The phosphorylation of PDK1 in S241 is catalyzed by an autophosphorylation reaction in trans.35
PDK1 kinase activity is therefore constitutively active, and the regulation of PDK1-activated signaling involves different mechanisms. The first mechanism was discovered investi-gating the steps involved in Akt T-loop phosphorylation in living cells. PDK1 is localized at the plasma membrane due to the interaction of its PH domain with the phosphoinositi-des PtdIns(3,4,5)P3, phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2 ], and PtdIns(4,5)P2, with the highest affinity toward the PI3K lipid products.33,36 Although PDK1
mem-brane localization has been largely investigated and the affinity of the PDK1-PH domain for the PI3K products suggested a potential PI3K-dependent PDK1 membrane translocation, this localization is still controversial. Indeed, the jury is still out on whether PDK1 translocates to the plasma membrane following growth factor stimulation, or it is constitutively localized to the plasma membrane. Nevertheless, it is well established that PDK1 membrane localization is essential for Akt phosphory-lation in Thr308 (Figure 2). PDK1 is constitutively associated in a homodimeric complex through PH domain interaction of two PDK1 monomers, and this interaction is important in the regulation of Akt phosphorylation.37
Many other kinases are known to be downstream of PDK1 and are attracting an increasing interest. Among these, serum glucocorticoid-dependent kinase (SGK), p70 ribosomal protein S6 kinases (S6K), p90 ribosomal protein S6 kinase (RSK), and atypical PKC isoforms are known to be direct targets of PDK1, which phosphorylates specific serine/
The mechanism of activation of these kinases differs from the Akt activation mechanism. PDK1 possesses a hydrophobic pocket, which is termed PDK1 interacting fragment pocket, and is essential for PDK1 interaction with the hydrophobic motif of the targeted protein kinases. Mutations within the PDK1 interacting fragment pocket abolish the binding of their subsequent phosphorylation and activation of PDK1 to PKC, S6K, and SGK1. The physiological role of PDK1 has been investigated in vivo in yeast, Drosophila melanogaster, and mice. These studies have shown that deletion of PDK1 is lethal, indicating that PDK1 is required for normal embryo development. PDK1–/– mice lack branchial arches, and have problems in neural crest specification and forebrain develop-ment, as well as several disruptions in the development of a functional circulatory system, which eventually causes death at the E9.5 embryonic stage. In order to study the role of PDK1 in development, hypomorphic mice for PDK1 were generated, in which the neomycin resistance gene is inserted between exons 2 and 3 of the PDK1 gene in order to reduce the expression of PDK1 by 80%–90% in all tissues.
These mice showed a decreased body size of 40%–50% compared to the wild type littermate, but no significant differences in the activation of AKT, S6K, and RSK were induced by insulin. Analysis of organs revealed that the difference in size is due to a decreased cell size rather than a reduction in cell number.
Specific function of PI3K/PDK1 in cancer
The PI3K pathway is one of the most frequently deregulated pathways in human malignancy; indeed, there are a variety of genetic abnormalities observed in this pathway in cancer, including activating and deactivating mutations, copy number changes, and posttranscriptional epigenetic irregularities. Among these, deactivating mutations in the gene encoding the tumor suppressor, PTEN, are among the most common.
Catalytic domain Pleckstrinhomology
Nuclear export sequence Tyr9
Ser25 Ser163 Ser241 Ser393/396 Ser410 Thr516
Tyr373/376
Figure 1 Schematic representation of PDK1 structure.
Figure 1.10: Schematic representation of PDK1 structure in its a) inactive and b) active
(modified from [19])
Conformational changes dependent activation was found to be a common feature for many kinases. In the past years the existence of an allosteric site, accessible only when the kinase turns in its catalytically inactive conformation, have been demonstrated. In the inactive form the Asp-‐Phe-‐Gly /DFG motif, located at the N terminus of the activation loop, is flipped “out” (DFG-‐out) relative to its conformation in the active
form (DFG-‐in) [20, 21].
1.4.2 Physiological role
PDK1 belongs to the ACG kinases family [18], composed of Serine and Threonine kinases, with a large sequence homology in the catalytic domain correlated to other kinases. Similar to other kinases of the family, PDK1 has a phosphorylation site that regulates its activation on Ser241. This site, situated in the activation loop (T-‐loop) located into the kinase domain, is phosphorylated in resting cells and not affected by growth factor stimulation. The phosphorylation of PDK1 in Ser241 is catalysed by an autophosphorylation reaction that occurs with a trans
mechanism [22], leading to a constitutive activation of the PDK1 kinase
activity.
PDK1 was originally discovered in 1997 as a PI3K dependent enzyme responsible for the phosphorylation of the Akt activation loop at the residue Thr308 [23]. PI3K activation by growth factors signalling induces growing levels of PIP2 and PIP3 and facilitates the interactions of Akt and
20
PDK1 with the plasma membrane through their respective PH domains, thus allowing PDK1 to phosphorylate Akt on Thr308 (Figure 1.11). This process was found to be dependent on the PIP3 concentration.
Moreover the conformational changes induced by the binding of PIP3 to
Akt also facilitates PDK1 phosphorylation [24].
Figure 1.11: Schematic representation of PDK1 dependent Akt (PKB) activation
(modified from [25]).
PDK1 is constitutively associated in a homodimeric complex through PH domain interaction of two PDK1 monomers, and this interaction is important in the regulation of Akt phosphorylation [4]. Also PDK1 membrane localization is essential for Akt phosphorylation, but, although it has been largely investigated and the affinity of the PDK1-‐PH domain for the PIP2 and PIP3 suggested a possible PI3K-‐dependent PDK1
membrane translocation, this localization is still controversial. Whether PDK1 translocates to the plasma membrane following growth factor stimulation, or it is constitutively localized to the plasma membrane it is still to establish.
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