Sonia Lain

Awakening guardian angels: drugging the p53 pathway

Currently, around 11 million people are living with a tumour that contains an inactivating mutation of TP53 (the human gene that encodes p53) and another 11 million have tumours in which the p53 pathway is partially abrogated through the inactivation of other signalling or effector components. The p53 pathway is therefore a prime target for new cancer drug development, and several original approaches to drug discovery that could have wide applications to drug development are being used. In one approach, molecules that activate p53 by blocking protein–protein interactions with MDM2 are in early clinical development. Remarkable progress has also been made in the development of p53-binding molecules that can rescue the function of certain p53 mutants. Finally, cell-based assays are being used to discover compounds that exploit the p53 pathway by either seeking targets and compounds that show synthetic lethality with TP53 mutations or by looking for non-genotoxic activators of the p53 response.

SUMO-1 MODIFICATION ACTIVATES THE TRANSCRIPTIONAL RESPONSE OF P53

The p53 tumour suppressor protein is regulated by ubiquitin‐mediated proteasomal degradation. In normal cells p53 is constitutively ubiquitylated by the Mdm2 ubiquitin ligase. When the p53 response is activated by stress signals p53 levels rise due to inhibition of this degradative pathway. Here we show that p53 is modified by the small ubiquitin‐like protein SUMO‐1 at a single site, K386, in the C‐terminus of the protein. Modification in vitro requires only SUMO‐1, the SUMO‐1 activating enzyme and ubc9. SUMO‐1 and ubiquitin modification do not compete for the same lysine acceptor sites in p53. Overexpression of SUMO‐1 activates the transcriptional activity of wild‐type p53, but not K386R p53 where the SUMO‐1 acceptor site has been mutated. The SUMO‐1 modification pathway therefore acts as a potential regulator of the p53 response and may represent a novel target for the development of therapeutically useful modulators of the p53 response.

Discovery, In Vivo Activity, and Mechanism of Action of a Small-Molecule p53 Activator

Summary We have carried out a cell-based screen aimed at discovering small molecules that activate p53 and have the potential to decrease tumor growth. Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6. Via a yeast genetic screen, biochemical assays, and target validation studies in mammalian cells, we show that tenovins act through inhibition of the protein-deacetylating activities of SirT1 and SirT2, two important members of the sirtuin family. Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents. This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.

Multiple C-Terminal Lysine Residues Target p53 for Ubiquitin-Proteasome-Mediated Degradation

ABSTRACT In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitin-activating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-to-arginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.

P53 abnormalities and outcomes in colorectal cancer: a systematic review

We performed a systematic review of studies that investigated the effect of abnormalities of the tumour suppressor gene p53 upon prognosis in patients with colorectal cancer. The methods used to assess p53 status were immunohistochemistry (IHC), indicating abnormal accumulation of p53, and sequence analysis, indicating presence of p53 mutations (mut). We identified 168 reports, with 241 comparisons of relevant end points and survival data on 18 766 patients. We found evidence of both publication bias and heterogeneity of results. Our analysis was hampered by variability in both the assessment of p53 status and the reporting of results. We used a trim and fill method to correct for publication bias and minimised heterogeneity by using well-defined clinical subgroups for the assessment of outcomes. Overall, patients with abnormal p53 were at increased risk of death: relative risk (RR) with IHC 1.32 (95% confidence interval (c.i.) 1.23–1.42) and with mutation analysis 1.31 (95% c.i. 1.19–1.45). The adverse impact of abnormal p53 was greater in patients with lower baseline risk of dying: good prognosis RR (mut) 1.63 (95% c.i. 1.40–1.90) and poor prognosis RR (mut) 1.04 (95% c.i. 0.91–1.19). We found no effect of abnormal p53 on outcome in patients treated with chemotherapy. Abnormal p53 was associated with failure of response to radiotherapy in patients with rectal cancer: RR (mut) 1.49 (95% c.i. 1.25–1.77).

Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide from p16CDKN2/INK4A

Abstract Background: The CDKN2/INK4A tumour suppressor gene is deleted or mutated in a large number of human cancers. Overexpression of its product, p16, has been shown to block the transition through the G 1 /S phase of the cell cycle in a pRb-dependent fashion by inhibiting the cyclin D-dependent kinases cdk4 and cdk6. Reconstitution of p16 function in transformed cells is therefore an attractive target for anti-cancer drug design. Results We have identified a 20-residue synthetic peptide — corresponding to amino acids 84–103 of p16 – that interacts with cdk4 and cdk6, and inhibits the in vitro phosphorylation of pRb mediated by cdk4–cyclin D1. The amino-acid residues of p16 important for its interaction with cdk4 and cdk6 and for the inhibition of pRb phosphorylation were defined by an alanine substitution series of peptides. In normal proliferating human HaCaT cells and in cells released from serum starvation, entry into S phase was blocked by the p16-derived peptide when it was coupled to a small peptide carrier molecule and applied directly to the tissue culture medium. This cell-cycle block was associated with an inhibition of pRb phosphorylation in vivo . Conclusion These results demonstrate that a p16-derived peptide can mediate three of the known functions of p16: firstly, it interacts with cdk4 and cdk6; secondly, it inhibits pRb phosphorylation in vitro and in vivo ; and thirdly, it blocks entry into S phase. The fact that one small synthetic peptide can enter the cells directly from the tissue culture medium to inhibit pRb phosphorylation and block cell-cycle progression makes this an attractive approach for future peptidometic drug design. Our results suggest a novel and exciting means by which the function of the p16 suppressor gene can be restored in human tumours.

Different effects of p14ARF on the levels of ubiquitinated p53 and Mdm2 in vivo.

Mdm2 has been shown to promote its own ubiquitination and the ubiquitination of the p53 tumour suppressor by virtue of its E3 ubiquitin ligase activity. This modification targets Mdm2 and p53 for degradation by the proteasome. The p14ARF tumour suppressor has been shown to inhibit degradation of p53 mediated by Mdm2. Several models have been proposed to explain this effect of p14ARF. Here we have compared the effects of p14ARF overexpression on the in vivo ubiquitination of p53 and Mdm2. We report that the inhibition of the Mdm2-mediated degradation of p53 by p14ARF is associated with a decrease in the proportion of ubiquitinated p53. The levels of polyubiquitinated p53 decreased preferentially compared to monoubiquitinated species. p14ARF overexpression increased the levels of Mdm2 but it did not reduce the overall levels of ubiquitinated Mdm2 in vivo. This is unexpected because p14ARF has been reported to inhibit the ubiquitination of Mdm2 in vitro. In addition we show that like p14ARF, the proteasome inhibitor MG132 can promote the accumulation of Mdm2 in the nucleolus and that this can occur in the absence of p14ARF expression. We also show that the mutation of the nucleolar localization signal of Mdm2 does not impair the overall ubiquitination of Mdm2 but is necessary for the effective polyubiquitination of p53. These studies reveal important differences in the regulation of the stability of p53 and of Mdm2.

p53-based Cancer Therapy

Inactivation of p53 functions is an almost universal feature of human cancer cells. This has spurred a tremendous effort to develop p53 based cancer therapies. Gene therapy using wild-type p53, delivered by adenovirus vectors, is now in widespread use in China. Other biologic approaches include the development of oncolytic viruses designed to replicate and kill only p53 defective cells and also the development of siRNA and antisense RNAs that activate p53 by inhibiting the function of the negative regulators Mdm2, MdmX, and HPV E6. The altered processing of p53 that occurs in tumor cells can elicit T-cell and B-cell responses to p53 that could be effective in eliminating cancer cells and p53 based vaccines are now in clinical trial. A number of small molecules that directly or indirectly activate the p53 response have also reached the clinic, of which the most advanced are the p53 mdm2 interaction inhibitors. Increased understanding of the p53 response is also allowing the development of powerful drug combinations that may increase the selectivity and safety of chemotherapy, by selective protection of normal cells and tissues.

Cell-cycle arrest and inhibition of Cdk4 activity by small peptides based on the carboxy-terminal domain of p21WAF1

BACKGROUND A common event in the development of human neoplasia is the inactivation of a damage-inducible cell-cycle checkpoint pathway regulated by p53. One approach to the restoration of this pathway is to mimic the activity of key downstream effectors. The cyclin-dependent kinase (Cdk) inhibitor p21(WAF1) is one such molecule, as it is a major mediator of the p53-dependent growth-arrest pathway, and can, by itself, mediate growth suppression. The primary function of the p21(WAF1) protein appears to be the inhibition of G1 cyclin-Cdk complexes. Thus, if we can identify the region(s) of p21(WAF1) that contain its inhibitor activity they may provide a template from which to develop novel anti-proliferative drugs for use in tumours with a defective p53 pathway. RESULTS We report on the discovery of small synthetic peptides based on the sequence of p21(WAF1) that bind to and inhibit cyclin D1-Cdk4. The peptides and the full-length protein are inhibitory at similar concentrations. A 20 amino-acid peptide based on the carboxy-terminal domain of p21(WAF1) inhibits Cdk4 activity with a concentration for half-maximal inhibition (l0.5) of 46 nM, and it is only four-fold less active than the full-length protein. The length of the peptide has been minimized and key hydrophobic residues forming the inhibitory domain have been defined. When introduced into cells, both a 20 amino-acid and truncated eight amino-acid peptide blocked phosphorylation of the retinoblastoma protein (pRb) and induced a potent G1/S growth arrest. These data support a physiological role for the carboxyl terminus of p21(WAF1) in the inhibition of Cdk4 activity. CONCLUSIONS We have discovered that a small peptide is sufficient to mimic p21(WAF1) function and inhibit the activity of a critical G1 cyclin-Cdk complex, preventing pRb phosphorylation and producing a G1 cell-cycle arrest in tissue culture cell systems. This makes cyclin D1-Cdk4 a realistic and exciting target for the design of novel synthetic compounds that can act as anti-proliferative agents in human cells.

Therapeutic exploitation of the p53 pathway

Analysis of the gene encoding p53 could serve to evaluate the effectiveness of a cancer treatment. Mutations in this gene occur in half of all human cancers, and regulation of the protein is defective in a variety of others. Novel strategies that exploit our knowledge of the function and regulation of p53 are being actively investigated. Strategies directed at treating tumours that have p53 mutations include gene therapy, viruses that only replicate in p53 deficient cells, and the search for small molecules that reactivate mutant p53. Potentiating the function of p53 in a non-genotoxic way in tumours that express wildtype protein can be achieved by inhibiting the expression and function of Mdm2 or viral oncoproteins.