by ecancer reporter Clare Sansom

The tumour suppressor protein p53 is a powerful regulator of cellular responses to stress.

It is known as the “guardian of the genome” and the gene that encodes it, TP53, is mutated or deleted in more than 50% of human tumours.

Activation of p53 can induce a number of different stress responses including apoptosis and cell cycle arrest.

Stimulation of p53 activity in tumours in which this protein is functional is an interesting and potentially important therapeutic strategy.

Several small-molecule p53 activators including Nutlin-3 have been developed in recent years.

However, p53 activation by these molecules most often results in reversible cell cycle arrest rather than apoptosis, which limits their use.

The molecular mechanisms that determine the fate of cells in which p53 is activated are not yet well understood. Joaquin Espinosa and his colleagues at the University of Colorado, Colorado, USA searched for genes that interact with p53 to modify cell fate under stress using a genome-wide short hairpin RNA (shRNA) screen.

They were looking specifically for genes that interact with p53 to promote cell cycle arrest and therefore cell survival. Inhibitors of the protein products of these genes might interact with Nutlin-3 and similar molecules to promote apoptosis.

This shRNA screen can identify genes that interact with activated p53 to induce apoptosis as cells in which these genes are silenced rapidly undergo apoptosis if treated with Nutlin-3. The researchers selected the top 500 of these “synthetic lethal with Nutlin” (SLN) genes and subjected them to pathway analysis to identify pathways that could be implicated in the promotion of p53-dependent apoptosis.

This analysis led them to focus on two such pathways, those controlled by the protein kinases ATM and MET.

Further tests confirmed that both these kinases themselves were strongly synthetic lethal, and this result indicated a possible mechanism for the known oncogenic activity of MET. Conversely, however, the identification of ATM as promoting cell survival in p53-activated cells was thought surprising as it is known to be an agonist of p53 activity during DNA damage.

The researchers then tested whether chemical inhibition of these kinases could promote apoptosis in cells treated with Nutlin-3. Human colon cancer cells with wild-type p53 were treated with varying concentrations of Nutlin-3 and the ATM-specific inhibitor KU-55933.

The combination of Nutlin-3 and KU-55933 resulted in the activation of the pro-apoptotic protease caspase-3 and in apoptosis, whereas neither drug induced apoptosis when given alone. Similar results were obtained in multi-cellular tumour spheroids (MCTs), a form of cell culture that is physiologically more similar to a tumour than typical two-dimensional cultures.

A similar test of MET activity was carried out using crizotinib, a small-molecule of this kinase that has been approved to treat non-small cell lung cancer. Crizotinib was shown to induce apoptosis in the same colon cancer cell line only when combined with Nutlin-3. The experiments were repeated in a number of cell lines and, while the results were fairly similar, the degree of synergy shown between Nutlin-3 and the kinase inhibitor varied between cell lines. Crucially, inhibition of these p53-interacting kinases did not affect the expression of a number of key p53 target genes.

These results identify the ATM and MET pathways as key regulators of cell fate after stress-induced activation of p53. Furthermore, they suggest that small molecule inhibitors of these kinases and perhaps other molecules in the same pathways may potentiate the activity of Nutlin-3 and other p53 activators in the large minority of tumours in which this protein is functional.

Reference

Sullivan, K.D., Padilla-Just, N., Henry, R.E., Porter, C.C., Kim, J., Tentler, J.J., Eckhardt, S.G., Tan, A.C., DeGregori, J. and Espinosa, J.M. (2012). ATM and MET kinases are synthetic lethal with nongenotoxic activation of p53. Nature Chemical Biology, published online ahead of print 3 June 2012. doi: 10.1038/nchembio.965