effectors of death receptor-mediated apoptosis, the caspases, have been successfully employed to promote glioma cell death.
Fig. 3.2 Mechanism of p53 control of cell cycle progres- sion. Amplification of MDM2 occurs in many tumors including glioma and is the most important inhibiting factors of p53. On the other hand, DNA-pk stabilizes p53 increasing its half-life and its intranuclear concentration. P53 activates p21. p21 is a potent cyclin-dependent kinase inhibitor that is able to bind to and inhibit the activity of cyclin-CDK2 or -CDK4 complexes, major regulators of cell cycle progression at G1. The com- plex cyclin D1-CDK4 can cause the hyper-phosphorylation of
the retinoblastoma protein (Rb) causing the progression of the cell cycle. When Rb is hypophosphorylated, it keeps locked the E2F-DP complex, and the cell remains in the G1 stage.
Hyperphosphorylation of Rb determines dissociation of E2F-DP from Rb and its activation. Unbound E2F activates factors like cyclins (e.g. cyclin E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases
dysfunctional in many cancer types. The name Rb derives from the retinoblastoma, the tumor in which this protein was identified. It is encoded by the RB1 gene that is located on chromosome 13 (13q14.1- q14.2). Because it is a tumor suppressor gene, both alleles must be mutated for the development of cancer.
Rb can be hyper-phosphorylated or hypophosphory- lated. Hyper-phosphorylation, due to raised activity of cyclin-cdk4, inhibits Rb. On the other hand, hypophos- phorylated Rb represents the active form, and it is able to inhibit cell cycle progression by binding and inhibiting the transcription factors of the E2F fam- ily. E2F is a transcription factor widely believed to integrate cell-cycle progression with the transcrip- tion apparatus through its cyclical interactions with important regulators of the cell cycle, such as Rb, cyclins and cyclin-dependent kinases. E2F exerts its role by binding specific DNA sequences to the pro- moter of the target genes. E2F family protein has a complex structure that presents some domains. A spe- cific domain is responsible for binding with a protein called DP, that functions as an inhibitor of E2F. As long as Rb is hypophosphorylated, it keeps locked E2F- DP complex, and the cell remains in the G1 stage.
Hyper-phosphorylation of Rb determines dissociation of E2F-DP from Rb and its activation. Unbound E2F
activates factors such as cyclins (e.g. cyclins E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases, and a molecule called prolif- erating cell nuclear antigen (PCNA) which favors the DNA replication.
P53 and p16/INKa4
In glioblastoma and anaplastic astrocytomas, a corre- lation between p53, MDM2, p16/INKa4, PTEN, and EGFR and survival rates seem to be present. Loss of p16/INKa4 is essential for maintenance of the trans- formed neoplastic phenotype (Shapiro et al., 1995).
This property of p16/INKa4 protein suggests that it is a tumor suppressor gene product that exerts its function in association with p53, both of which inhibit cyclin- dependent kinases involved in the Rb pathway. It is probable that cells unable to produce p16/INKa4 pro- tein may be vulnerable to neoplastic transformation.
p16/INKa4 protein is a potent inhibitor of cdk-4 that blocks cdk4-mediated phosphorylation of the tumor suppressor Rb protein, allowing Rb-mediated growth suppression (Fig. 3.2). The cdk4/cyclin D1 com- plex phosphorylates the Rb protein, thereby inducing release of the E2F transcription factor that activates
genes involved in the G1 to S transition. p16/INKa4 binds to CDK4, inhibits the cdk4/cyclin D1 complex, and thus inhibits the G1 to S transition. Thus, loss of normal RB1 function may result from altered expres- sion of any of the RB1, p16/INKa4, or Cdk4 gene. This means that pathways of both p53 and p16/INKa4 have a convergence of activity on the pRB protein.
P53 and PTEN
A broad relationship between p53 and PTEN (phos- phatase and tensin homolog deleted on chromosome 10) has been described. PTEN is a tumor suppres- sor gene whose mutation was found in various types of sporadic tumors, and was originally described in malignant gliomas. The promoter of PTEN has a p53 binding site. In fact, wild-type p53 can promote PTEN transcription, whereas TGF-β down-regulates it. The protein encoded by PTEN is involved in the regulation of many important cellular functions, such as cell- cycle progression, cell migration and spreading, cell growth, and apoptosis. As p53 modulates transcription of PTEN gene, the protein protects p53 from MDM2- mediated degradation through a PI3K/Akt pathway.
In fact, there is a positive feedback between p53 and PTEN which aims to control the cellular response to stress, DNA damage, and cancer.
PTEN has a phosphatase activity against phospho- inositide substrates; it dephosphorylates, with high activity, the 3-OH position of the inositol ring of phosphatidylinositol phosphates, in particular of phos- phatidylinositol 3-phosphate (PIP3), thereby acting as the counterpart of phosphoinositide-3-kinase (PI3K).
Actually, PI3K phosphorylates phosphatidynositol- 4,5-bisphosphate to the respective 3-phosphate (PIP3), which functions as a second messenger molecule that is important for the activation of protein kinase B/Akt (for reviews, see Vanhaesebroeck and Alessi,2000). In addition, PIP3 facilitates the translocation of Akt to the plasma membrane and activates PDK1, which in turn phosphorylates Akt on threonine 308 within the kinase domain. Activated Akt in turn can phosphorylate a variety of substrates and thereby regulates important cellular processes, including cell-cycle progression, cell growth, cell survival, cell motility and adhesion, translation of mRNA into protein, glucose metabolism, and angiogenesis. PTEN may influence the activity
of several cellular signalling pathways other than the PI3K/Akt pathway. For example, the lipid phosphatase activity of PTEN may also contribute to the inhibition of Ras and MAPK pathway activation by EGF.
In gliomas, PTEN mutations are preferentially found in glioblastomas, with reported frequencies of up to 40%. PTEN mutations are frequent in primary (de novo) glioblastomas, less frequent (<10% of the cases) in secondary glioblastomas, namely in glioblas- tomas that have progressed from pre-existing lower grade gliomas. PTEN showed to play an important role in maintaining the cellular susceptibility to apop- totic stimuli. Gene transfer into U-87 MG glioma cells that lack wild-type PTEN renders the cells susceptible to apoptosis. In addition, PTEN gene transfer sen- sitizes glioma cells for apoptosis when irradiated or treated with FasL. The regulatory function of PTEN on apoptosis is dependent on PI3K/Akt signaling.
PTEN wild-type cells overexpressing mutant (consti- tutively active) Akt are resistant to multiple apoptotic stimuli, as are PTEN-mutant cells. Akt itself is a ser- ine/threonine kinase that can phosphorylate several apoptosis-associated proteins, including the proapop- totic factor BAD. Upon phosphorylation, BAD can no longer function as a proapoptotic molecule and dissociates from BclXL or Bcl-2, leading to a rela- tive increase in the level of anti-apoptotic proteins.
Although the role of BAD in Akt-mediated inhibi- tion of apoptosis is well characterized, it is likely that PTEN/PI3K/Akt signaling may additionally influ- ence apoptosis using other targets, such as glyco- gen synthase kinase 3, p70S6k, or IkB/NF-κB. There are also recent evidence of the involvement of the PTEN/PI3K/Akt signaling pathway in the control of death receptor- and drug-induced apoptosis. Recently, Opel et al. (2008) demonstrated that inhibition of PI3K by LY294002 sensitizes wild-type and mutant PTEN glioblastoma cells to both death receptor- and chemotherapy-induced apoptosis. LY294002 sig- nificantly enhances apoptosis triggered by TRAIL, agonistic anti-CD95 antibodies, or several anticancer drugs (i.e., doxorubicin, etoposide, and vincristine) in a highly synergistic manner. In addition, LY294002 cooperates with TRAIL or doxorubicin to suppress colony formation, thus also showing a strong effect on long-term survival. Similarly, genetic knockdown of PI3K subunits p110αand/or p110βby RNA interfer- ence (RNAi) primes glioblastoma cells for TRAIL- or doxorubicin-mediated apoptosis. Analysis of apoptosis
pathways revealed that PI3K inhibition acts in concert with TRAIL or doxorubicin to trigger mitochondrial membrane permeabilization, caspase activation, and caspase-dependent apoptosis.