Chromosome 10q is frequently lost (50-80%) in prostatic lesions. PTEN is located within this locus (10q23) and is a well-known negative regulator of the PI3K/Akt signal transduction cascade. The PI3K/Akt signaling pathway is known for its role in mediating cell survival, as well as cell cycle progression and neoplastic transformation.17
Consequently, it would stand to reason that mutation of PTEN would initiate heightened activation of this cascade, which could then confer a number of cancer-like properties to the cell. Many of these, particularly cellular survival, are hallmark features of CaP tumor cells.
Phosphatidylinositol-3-kinase (PI3K) is a heterodimeric protein with an 85-kDa regulatory subunit and a 110-kDacatalytic subunit. PI3K serves to phosphorylate a series of membrane phospholipids including PtdIns(4)P and PtdIns(4,5)P2
, catalyzing the transfer of ATP-derived phosphate to the D-3 position of the inositol ring of membrane phosphoinositides, thereby forming the second messenger lipids PtdIns(3,4)P2
and PtdIns(3,4,5)P3. Most often, PI3K is activated via the binding of a ligand to its cognate receptor, whereby p85 associates with phosphorylated tyrosine residues on the receptor via an SH2 (Src-homology 2) domain. After association with the receptor, the p110 catalytic subunit then transfers phosphate groups to the aforementioned membrane phospholipids. It is these lipids, specifically PtdIns(3,4,5)P3
, that attract a series of kinases to the plasma membrane thereby initiating the signaling cascade.18
An overview of the PI3K/PTEN/Akt/mTOR pathway is presented in .
Figure 1 Overview of PI3K/PTEN/Akt/mTOR Pathway. The PI3K/PTEN/Akt/mTOR pathway is regulated by Ras as well as various kinases. The PI3K/PTEN/Akt/mTOR pathway is also activated after receptor ligation. The PTEN phosphatase (black octagon) inhibits activation of (more ...)
Downstream of PI3K is the primary effector molecule of the PI3K signaling cascade, Akt/PKB (protein kinase B). Akt was originally discovered as the cellular homologue of the transforming retrovirus AKT8 and as a kinase with properties similar to protein kinases A and C.19,20
Akt contains an amino-terminal pleckstrin homology (PH) domain that serves to target the protein to the membrane for activation.21-23
Within its central region, Akt has a large kinase domain and is flanked on the carboxy-terminus by hydrophobic and proline-rich regions.24,25
Akt is activated via phosphorylation of two residues: T308, S473. While it is generally agreed upon that maximal activation of Akt requires phosphorylation of both of these residues, there are conflicting reports regarding the relative importance of S473 and T308 for initial activation of Akt. It was originally thought that either of these residues could be phosphorylated to initiate Akt activity and that phosphorylation of one residue was not necessary for phosphorylation of the other to occur.26
More recent evidence suggests that phosphorylation of T308 is absolutely essential for phosphorylation of S473.27,28
Each of these studies was carried out in the HEK293 cell line, which further compounds the issue because the differences cannot be attributed to cell type.
The phosphotidylinositide-dependent kinases (PDKs) are responsible for activation of Akt. PDK1 is the kinase responsible for phosphorylation of T308,29
while the actual kinase that phosphorylates S473 (PDK2) remains controversial. Some report that phosphorylation of T308 triggers autophosphorylation of S473,30
while others hypothesize that PDK2 is the integrin-linked kinase (ILK).31
However, it is now believed that ILK may facilitate phosphor-ylation of S473 indirectly, perhaps by acting as a scaffolding protein.32
Akt can also be phosphorylated by the mTOR complex called Rictor.33
Phosphorylation of Akt is complicated as it can be phosphorylated by a complex which lies downstream of itself. Moreover, it can be dephosphorylated by p70S6K
which also lies downstream of Akt.34
Once activated on either/both of these residues, Akt leaves the cell membrane to phosphorylate intracellular substrates.
After activation, Akt is able to translocate to the nucleus35
where it affects the activity of a number of transcriptional regulators. Cyclic-AMP response element binding protein (CREB),36
NFκB (via Iκ-K),40,41
and the forkhead transcription factors42-50
are all either direct or indirect substrates of Akt and each can promote either cellular proliferation or survival. Aside from transcription factors, Akt is able to target a number of other molecules to affect the survival state of the cell including caspase-9,51
the pro-apoptotic molecule BAD,52,53
and glycogen-synthase kinase-3β
When these targets are phosphorylated by Akt, they may either be activated or inactivated but the end result is to promote survival of the cell.
Negative regulation of the PI3K pathway is primarily accomplished through the action of the PTEN tumor suppressor protein. PTEN
encodes a lipid and protein phosphatase whose primary lipid substrate is PtdIns(3,4,5)P3
. The protein substrate(s) of PTEN
are more varied, including focal adhesion kinase (FAK), the Shc exchange protein and the transcriptional regulators ETS-2,55
PTEN also may negatively regulate the activation of the platelet-derived growth factor (PDGF) receptor.57
PTEN has four primary structural domains. On the amino terminus is the lipid and protein phosphatase domain, which is flanked adjacent to the C2 domain that is responsible for lipid binding and membrane localization. Next are two PEST domains which regulate protein stability. Lastly, PTEN has a PDZ domain, which helps facilitate protein-protein interactions. Mutations within the phosphatase domain have been reported to nullify the endogenous function of PTEN.58-60
As previously mentioned, loss of PTEN function in advanced CaP is quite common (50-80% of patients).61,62
As a consequence, this results in an overabundance of lipid second messengers [PtdIns(3,4,5)P3
], which can cause constitutive activation of PH domain-containing proteins including Akt. It is for this reason that Akt is found to be highly-activated in advanced cases of CaP.63,64
Aberrant Akt activation is able to elicit the pro-survival properties observed in CaP cells through a number of mechanisms, described hereafter. Hence inhibiting Akt or restoring PTEN activities are potential therapeutic targets in prostate cancer.
is a central mediator of cell cycle progression whose role as a cyclin-dependent kinase (CDK) inhibitor is lost in many cases of advanced CaPs.65-69
The role of p27Kip1
in preventing prostatic disease is bolstered by evidence which shows that p27Kip1
-null mice quickly develop prostatic hyperplasia.70
Akt appears to affect p27Kip1
expression via the intermediate molecule, FKHR. FKHR (FOXO1) is a member of the forkhead/FoxO family of transcription factors, which are known to stimulate transcription of p27Kip1
Upon activation, Akt phosphorylates FKHR causing its inactivation and subsequent downregulation of p27Kip1
. Further evidence has shown that constitutively-active Akt may be able to decrease the half-life of p27Kip1
This series of events leads to a phenotype whereby CaP cells have less restriction on cell cycle progression, thereby promoting unregulated cell division. Supporting these observations are other studies which show that as CaP progresses from an androgen-dependent to -independent state, p27Kip1
levels are drastically diminished.74,75
Akt regulates the apoptotic response to a variety of stimuli via its ability to interact with a number of key players in the apoptotic process. First, Akt can directly phosphorylate BAD on S136,76
causing its inactivation and inability to interact with anti-apoptotic members of the Bcl-2 family of proteins (Bcl-2, Bcl-XL
Next, activated Akt can independently inhibit the release of cytochrome c from the mitochondria, which is a potent activator of the apoptotic caspase cascade.65
Akt also is capable of phosphorylating procaspase-9, preventing its cleavage into the pro-apoptotic caspase-9 initiator of programmed cell death.51
Lastly, the Akt target, FKHR is capable of upregulating Fas ligand and Bim, two very important molecules that are potent inducers of apoptosis; however, when inactivated by Akt, FKHR is localized to the cytosol where it is unable to augment expression of these genes.49,79
Akt can also phosphorylate Bim which inhibits its proapoptotic activity. In concert, these events caused by Akt activation would appear to greatly affect the survival status of the cell.
Akt also plays significant roles in protein translation, particularly by regulating those proteins involved in growth and survival. A specific target is mTOR (mammalian target of rapamycin) which is able to induce phosphorylation of eIF-4E binding protein-1 (4E-BP1). After the appropriate phosphorylation events, 4E-BP1 disassociates from the mRNA cap-binding protein eIF-4E, which allows eIF-4E to interact with the eIF-4E translation initiation complex to initiate protein synthesis. Another target of Akt is p70S6K
which is a well-known enhancer of protein synthesis.80-84
mTOR has been shown to be critically important in autophagy,85-87
a mechanism of cell death which will be discussed below.
Messenger RNAs differ in their ability to be translated; the length and sequence of the 5' UTR largely dictates the efficiency with which an mRNA transcript will be translated. Most mRNAs contain short, unstructured GC-poor 5' UTRs and are efficiently translated. In contrast, protooncogene and growth factor mRNAs are characterized by long, GC-rich sequences in the 5' UTR, which can often hinder the ability of the eIF-4E complex to efficiently scan and initiate translation at the start codon. Consequently, under normal circumstances these mRNAs are not efficiently translated.88,89
However, upon Akt-mediated activation of mTOR, these latter mRNAs are highly and disproportionately translated; several key proteins that are overexpressed as a consequence of this event include c-myc,90-96
Cyclin D1 has been reported to be overexpressed in CaP xenografts and metastases,100,101
while early stage prostatic lesions possess much lower levels of the protein.102
A number of reports support the notion that mTOR signaling is a prominent feature of CaP progression, as recurrent tumors have altered expression of a number of molecular targets of rapamycin.103-107
Hence mTOR inhibitors such as rapamycin may be effective in prostate cancer therapy.