Tumor Suppressor Genes

Review of p53 (html) (pdf)

The formation of tumors is a multistep process requiring progressive accumulation of genetic alterations. Hence, it is becoming clear why loss of p53 plays such a pivotal role in human cancer. Accumulating the half-dozen or so mutations necessary for a cell to become carcinogenic requires genetic instability--a consequence of p53 loss. Another important step is relief of the apoptotic barrier that prevents uncontrolled growth--a second consequence of p53 loss.

The tumor suppressor p53 plays a critical role in preventing human cancer formation. In response to a variety of stress signals, often associated with the progression of neoplastic diseases, p53 becomes activated and induces cell cycle arrest and/or programmed cell death (apoptosis). By eliminating damaged and potentially dangerous cells that might otherwise become cancerous, p53 suppresses tumor formation. p53 has been called the "gatekeeper of the genome."

p53 is a transcription factor

The p53 tumor suppessor protein is a tetrameric phosphoprotein (Figure CA5). The 393-residue polypeptide contains four functional domains. The proline-rich, acidic, N-terminal domain (residues 1-43) is involved in transcriptional activation. This is also the site where Mdm2 binds. Mdm2 binding is prevented by the phosphorylation of serine residues at positions 15 and 20. The large, central core domain (residues 100-300) is involved in DNA binding, and is the location of almost all oncogenic p53 mutations. The oligomerization domain (residues 320-360) contains nuclear localization signals and is involved in p53 tetramerization. The basic C-terminal domain (residues 364-393) is a negative regulatory domain that can inhibit sequence-specific DNA binding by the core domain. Covalent modifications adding acetyl groups to lysine residues within this domain play a role in activating latent p53.

p53 is regulated by specific proteolytic degradation

In unstressed cells, p53 is latent and is maintained at low levels by targeted degradation mediated by its negative regulator, Mdm2 (Figure CA6). The critical role of Mdm2 in regulating p53 is best illustrated by a study carried out in mice where inactivation of p53 was shown to completely rescue the embryonic lethality caused by the loss of Mdm2 function (Mice lacking p53 are viable, but have increased incidence of cancer). Mdm2 counteracts p53 tumor suppressor activity by physically binding to p53 and suppressing its transcriptional activity. Mdm2 also functions as the p53 ubiquitin ligase and triggers its degradation. This latter activity requires the Ring finger domain located at the C-terminus of Mdm2. The MDM2 gene itself is under transcriptional control by p53.

Three major pathways lead to p53 activation

The activation and stabilization of p53 are thought to be mediated by specific protein modifications, with phosphorylations being the major events (Figure CA7). Although the exact functions of specific phosphorylation events remain controversial, evidence indicates that they probably contribute to both the stabilization and activation of p53. For example, DNA-damaging agents activate phosphorylation at serine (Ser) 15, likely by a family of protein kinases including ATM and ATR, and Ser20 by the Chk2 kinase. These phosphorylation events are believed to contribute to p53 stabilization by preventing the binding of Mdm2 and rendering p53 more resistant to Mdm2.

In addition to potentially regulating Mdm2 binding, phosphorylation was also shown to modulate the transcriptional activity of p53. For example, phosphorylation at Ser15 stimulates p53 interaction with its transcriptional co-activators p300 and CBP, and a mutation that eliminates this phosphorylation leads to p53 transcriptional defects.

Inappropriate expression viral or cellular oncogenes, such as ras or myc, leads to p53 activation through a p14ARF-dependent pathway. p14ARF functions, at least in part, by binding to Mdm2 and neutralizing its activity. p14ARFinhibits the p53 ubiquitin ligase activity of Mdm2. Because the ubiquitin ligase activity of Mdm2 appears to be essential for the degradation of p53, it is possible that by directly binding and inactivating Mdm2, p14ARF bypasses the need for phosphorylation in p53 activation.

p53 activation also requires acetylation

Another potential mechanism that may play a critical role in p53 activation is acetylation. Multiple lysine (K) residues in p53 are acetylated by p300 and its family member CBP or by P/CAF. In vivo studies show that some of these sites are acetylated in response to DNA-damaging agents, demonstrating that acetylation is a bona fide modification for p53. Acetylation stimulates p53 DNA binding activity in vitro. p300 and CBP were originally discovered as transcriptional co-activators that play critical roles in integrating multiple signal-dependent transcription events. In vivo, genetic experiments have clearly demonstrated essential roles for p300 and CBP in normal embryonic development. More recent analyses have indicated that p300 and CBP may have specific roles in tumor suppression pathways. p300 mutations were recently found in many types of tumors and mutation of human CBP causes RubinsteinTaybi syndrome (RTS), which leads to an increased risk of cancers. Interestingly, many of the p300 mutations identified from tumors actually result in the loss of acetyltransferase activity, suggesting that the ability of p300 and CBP to acetylate one or more cellular proteins may be critical for their functions in growth control. The fact that p300 and CBP play important roles in p53 transcriptional activity suggests that p53 might be a critical substrate of p300/CBP in mediating tumor suppression.

Figure CA8 summarizes the many ways in which p53 may malfunction in human cancers.

p53-dependent transcription of target genes leads to cell cycle arrest or cell death (apoptosis)

The p53-dependent transcription of target genes responds to a diverse range of cellular signals that affect cell proliferation and DNA integrity checkpoints. In undamaged cells that are dividing normally, p53 is highly unstable, with a half-life measured in minutes. After DNA damage induced by ionizing radiation (which is the only type of damage discussed here) the half-life increases significantly, leading to accumulation of p53 and transcription of target genes such as p21WAF1/CIP1 and BAX. The outcome of this increased transcription depends on the type of cell but usually is manifest as a very prolonged (possibly irreversible) G1 arrest or apoptosis (Figure CA9).

It is worth remembering that the response of p53 to DNA damage is not required for cell survival. However, the survival of the whole organism is affected because activation of p53 after DNA damage results in removal of potentially mutant cells from the population by inducing them to enter prolonged arrest or apoptosis. Loss of p53 does not result in cellular radiation sensitivity; its loss actually increases survival rates in many cell types because individual cells escape arrest or apoptosis. Instead, loss of p53 results in decreased genome stability, not because of loss of transient checkpoint controls (these remain substantially intact in cells that lack p53), but because loss of p53 creates an environment that is permissive for genome instability--that is, more damaged cells with chromosome aberrations and mutations survive and propagate. Thus, the main role of p53 in genome stability is the removal of problematic cells from the population when other systems fail.

Defects in cell cycle control can lead to cancer. All eucaryotic cells have cyclins and cyclin-dependent protein kinases (CDKs) that mediate the transition from one stage of the cell cycle to the next. In addition to CDKs, all cells have checkpoint systems that prevent CDKs from initiating the next cell cycle phase until critical processes in the current phase are complete. One major checkpoint mechanism is orchestrated by a set of proteins called cyclin-dependent kinase inhibitors, which bind and inactivate CDKs. The start of S phase (when DNA is synthesized) is delayed in eukaryotes when G1 cells are UV-irradiated. In cells lacking p53, the delay in S phase does not occur, and cells enter S phase with damaged DNA. If wildtype p53 is present, the cells enter S phase only after the damage is repaired, or, if the damage is too severe, the cells undergo apoptosis. The discovery that p53 stimulates transcription of the cyclin-dependent kinase inhibitor gene p21WAF1/CIP1, which encodes a G1 cyclin/CDK inhibitor, in response to DNA damage provides a molecular understanding of the basis for S phase delay. The p21WAF1/CIP1 upstream region contains a p53 binding site (the consensus target site is RRRC(A/T)(T/A)GYYY), and accumulation of p21WAF1/CIP1 activity and cell cycle arrest in irradiated cells requires wildtype p53.

There are several potential mediators of p53-induced apoptosis. The Bax protein is an apoptosis-inducing member of the Bcl-2 protein family. Transcription of the BAX gene is directly activated by p53-binding sites in the regulatory region of the gene. Bax is located in mitochondria. When overexpressed, Bax induces apoptosis.