WEHI team exposes two new anti-cancer targets

Cancer researchers in Melbourne and Innsbruck hunting for new options to treat cancer have flushed two promising new therapeutic targets from the genetic thickets of the p53-mediated apoptosis pathway: Puma and Noxa.

As well as being potentially prime targets for new anti-cancer cancer therapies, Puma and Noxa could also be candidates for designer drugs to reduce damage to the central nervous system and heart after stroke, heart attack and physical injury.

Collaborating research teams led by Dr Andreas Strasser at the Walter and Eliza Medical Research Institute (WEHI) in Melbourne, and Dr Andreas Villunger, at the University of Innsbruck in Austria, announced their discoveries this week in the international journal Science. Villunger worked in Strasser's laboratory at the Hall Institute before returning to Austria 12 months ago.

They have shown that Puma and Noxa are both critical for apoptosis, or programmed cell suicide.

Strasser said p53, the archetypal tumor-suppressor gene, was probably the most important gene in human cancer. Sometimes known as the 'guardian of the genome', it has a critical role in preventing cancer. It initiates cell-cycle arrest and apoptosis in genetically damaged or injured cells, and is activated in normal cells by genomic damage, stress, injury or early events in the process of tumour development.

Many chemotherapeutic drugs and radiation therapy kill cancerous cells by causing genetic damage that activates the p53 cell-suicide pathway.

In about 50 per cent of human cancers, p53 is itself inactivated by mutation; mutant cells lacking p53 protection can escape growth arrest and apoptosis and this allows them to become cancerous.

Strasser said the main mechanism by which p53 triggers cell cycle arrest of cells was known -- it activates another gene, P21, that encodes a cyclin-dependent kinase inhibitor, which prevents the cell from initiating DNA replication.

"But it has not been clear how p53 kills cells when it is activated," Strasser said. "P53 is a transcription factor, with 16 known target genes that have been proposed to be involved in apoptosis. There has also been speculation that p53 can kill cells directly by attacking the mitochondria.

Cascade of events

P53 initiates a cascade of genetic events that switches off the pro-survival gene Bcl-2 (and/or its homologues). Switching off Bcl-2 activates caspase enzymes that are ultimately responsible for killing the damaged cell. But Strasser said little was known about what happened between p53 switching on, and Bcl-2 switching off.

Strasser and his colleagues did not discover Noxa or Puma, but were very interested in them because both encode proteins from the BH3-only family.

The WEHI team, and research teams in the US, discovered the BH3-only protein family,which is essential for initiation of programmed cell death. The gene family is ancient -- it is present in the worm Caenorhabditis elegans.

Mammals have at least eight BH3-only genes and Strasser's team suspected one or more of them might act as links between p53 and Bcl-2 in apoptosis signalling.

Intriguingly, Strasser's team and others found evidence that has implicated the BH3-only proteins in programmed cell death induced by stimuli that do not activate p53.

"Noxa and Puma had excellent credentials as regulators of cell death and survival in cells suffering DNA damage because both genes are up-regulated by p53," Strasser said.

Strasser's team set out to create knockout mice for all eight BH3-only proteins, including Puma and Nox. The team now has knockouts for six of the eight genes.

"Because there are so many of them, there's probably some functional overlap, so we're now also making double- and triple-knockout mice because we believe that it will provide interesting insights into the control of cell death and cancer," Strasser said.

"Many others thought the same, and there was a huge race to knock out these genes. We were equal first with a Japanese team for a Noxa knockout, but we won the Puma knockout race."

Noxa-knockout mice exhibit no detectable abnormality in apoptosis function in many cell types, but their embryonic fibroblasts are abnormally resistant to DNA-damaging chemotherapy agents and radiation that would normally induce p53-mediated apoptosis.

Strasser said Puma was even more interesting. "The apoptosis defect we have observed in Noxa-knockout fibroblasts is there in the Puma knockout as well. But in the Puma knockout, many other cell types, including all lymphoid cell types we tested, are also resistant to chemotherapy agents and gamma radiation."

Strasser said this suggested Puma might be a good target for a generalised therapy for all leukaemias and lymphomas and perhaps other tumours as well -- provided its activity could be restricted to cancerous cells.

"Another intriguing thing is that lymphoid cells in the Puma knockout mouse are not only resistant to p53-induced cell-death stimuli, but also to certain death stimuli that are not mediated by p53," he said.

Activating apoptosis

Strasser said nothing was known about how phorbol esters, or the glucocorticoids used to treat certain childhood leukaemias, killed cells. Significantly, Puma-knockout lymphoid cells are also highly resistant to these agents.

Strasser said oxygen deprivation (anoxia) was known to activate apoptosis via a p53-dependent pathway. "It will be interesting to test if this pathway requires Puma or Noxa or both.

"It is possible that pathological cell death caused by stroke, ischaemic heart attack, and perhaps by certain types of injuries to the nervous system, such as near-drowning and trauma, are mediated by p53, possibly by activating Puma or Noxa.

"If this turns out to be the case, then a drug that suppresses Puma or Noxa activity might minimise nerve and tissue damage in these pathological conditions.

While Puma and Noxa are primarily activated within the p53 pathway, they may be directly activated by stimuli that do not engage p53. The Melbourne-Innsbruck team is now attempting to determine whether they function as tumour-suppressors in their own right.

"We are crossing Puma- and Noxa-knockout mice with mice that over-express oncogenes," he said. "We're pretty sure we're going to see accelerated tumorigenesis."