Feature: New pathways for cancer therapy

This feature appeared in the September/October 2011 issue of Australian Life Scientist. To subscribe to the magazine, go here.

Associate Professor Stuart Pitson has not always been the enigmatic-lipid type of guy. Indeed, his initial expertise was in the more basic biochemical field of enzymology, starting with a PhD at La Trobe University in Victoria studying fungal enzymes.

His first postdoctoral stint in the Netherlands was also centred around fungal enzymes, while his second postdoc back in Australia at the University of New South Wales saw Pitson focus on characterising a set of enzymes from the infamous gastric bacteria, Helicobacter pylori.

After all those microbes, Pitson decided on a change of scientific scenery. He had become interested in the area of lipid signalling and predominantly in sphingosine kinase-related pathways. “The field then was really just based on phenomenology,” he says.

“No-one really understood how this enzyme, sphingosine kinase [SK], worked. None of the relevant enzymes had been cloned and only a bit of basic cell biology had been done. It presented a great opportunity to apply my expertise and take the field a lot further by combining molecular and cell biology with some ‘real’ enzymology.”

Pitson is currently pursuing this vision as an NHMRC Senior Research Fellow and Head of the Molecular Signalling Laboratory at the Centre for Cancer Biology, SA Pathology, in Adelaide. His group researches sphingolipid-mediated cell signalling pathways, and how they contribute to cancer and other diseases, with a particular focus on the key enzyme, SK.

Lipid molecules like the sphingolipids have long been recognised as important players in the cell, but they have also always been quite tricky characters to pin down in terms of the genes involved, regulation and functional significance.

However, over the last decade or so, more focus has been placed on lipid signalling with the realisation of its role in a host of pathological processes including inflammation and cancer.

Centre forward on the sphingolipid-signalling field of play is SK. This enzyme is a conserved and ubiquitiously expressed lipid kinase that catalyses the phosphorylation of sphingosine to form sphingosine-1-phosphate (S1P), a messenger protein involved in diverse intra- and extra-cellular processes. There are two main isoforms, but the most widely characterised and studied is SK1.

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Of interest to Pitson were early findings that elevated SK activity in the cell promotes cell proliferation, protects cells from apoptosis (programmed cell death) and, most importantly, induces neoplastic transformation (normal cell changing into a cancer cell).

This suggested a role for SK in tumour biology, which was supported by subsequent findings of upregulated SK in many cancer types and inhibition of tumour growth in animals when SK was blocked by inhibition or genetic manipulation.

All of this pointed to SK as a potential target in cancer therapy. The problem was that very little was known about how this enzyme worked or how it was regulated in the cell, which is where Pitson enters the story.

“When I joined the group in Adelaide in 1998 to work on SK, the Centre already had a strong cell biology focus led by Mathew Vadas. So, it seemed like a perfect fit for me to come in and add my skills in enzymology and biochemistry to the mix.”

History proved Pitson correct, and within two years he was the first to clone the human gene for SK1. Not long after that, Pitson established his own group within the Institute and ultimately went on to expand the sphingosine signalling field.

Filling in the holes

According to Pitson, the field exploded early in the new millennium with the discovery of the cell surface receptors for sphingosine-1-phosphate, the main product of SK activity. “This really put SK on the map as a bone fide signalling molecule and gave all the data about the enzyme’s effects on cell growth, survival and neoplastic transformation some substance.”

Suddenly it seemed like the whole world of lipid biologists were working on these cell surface receptors. However, Pitson remained focussed on his own favourite mystery. “I was particularly keen by this time to understand how this whole SK system worked in the context of trying to target cancer.

The SK knockout mouse had recently been made and showed that getting rid of all SK is embryonically lethal, confirming its functional importance. If we could nut out the exact regulatory mechanisms, we could potentially control this enzyme in situations like cancer.”

Pitson’s initial idea was that direct inhibitors of the SK enzyme might have too many detrimental effects, and taking out a molecule that is crucial for so many processes is often a double-edged sword.

“So our idea was to specifically target just the activation mechanism for SK, because some of our earlier work revealed that activation of SK is critical to its tumorigenic signalling.” Easy! They just first had to work out how SK activation actually works, so that is precisely what they did.

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It turns out that SK activation works a little bit differently from most other protein kinase enzymes. Rather than being either completely on or completely off, SK already has intrinsic catalytic activity and works by getting a little boost of activation.

Pitson’s group discovered that the enzyme activity of SK can be increased to the required levels via phosphorylation by other protein kinases in the cell – specifically the pivotal MAPK/ERK family members – offering a more complex level of control. Even more strikingly, mutation of the activation phosphorylation site in SK blocked its role in cell proliferation and survival. And it blocked tumour growth.

More recently, Pitson’s team also described another key regulatory switch for SK that seems particularly important for its role in cancer . “We found that as well as the MAPK/ERK phosphorylation increasing the activity of SK, it also results in a translocation from where it normally hangs out in the cytoplasm to the plasma membrane. Furthermore, we could show that this shift in cellular location is absolutely critical for SK’s oncogenic signalling.”

Pitson’s team then did the reverse experiment and blocked this translocation of SK, with the very exciting result that, without the shift in location, the enzyme showed no oncogenic signalling. It was still active in its intracellular site, and able to go about most of its normal business but, if not at the cell surface, the cancer-promoting activity of SK was just not there.

Then just last year, the group finally nailed exactly how their favourite enzyme makes its journey to the plasma membrane: by hitching a ride with a small calcium-myristoyl switch protein called CIB1.

Although the fine details of SK’s transport arrangements are still being worked out, Pitson believes that the CIB1 protein carries the SK to the cell surface in a process that is both calcium sensitive and regulated by CIB1’s myristoyl group and SK phosphorylation.

The end point is that SK gets deposited at the right place on the membrane and in the right conformation to stay there and do its job in cell proliferation.

“On the back of these findings we are now also looking at both CIB1 and the SK-CIB1 interaction as potential targets for cancer therapy. Rather than blocking the activity of the enzyme itself, we might only need to stop it getting to where it does its job to drive cancer.” This work is now the focus of one of Pitson’s current NHMRC grants.

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Targeting the targets

Along with many other groups around the globe, Pitson’s team is also trying to develop direct SK inhibitors because there is some evidence that such agents might still work to quell tumour growth despite the probable side effects.

None of the SK inhibitors floating around have yet made it into clinical trials, although Pitson reports that some have showed efficacy in animal studies.

“For our part, we have set up a program in collaboration with Michael Parker at St. Vincent's Institute in Melbourne trying to crystallise the SK protein and develop inhibitors that way, although this is proving quite difficult.

“Being a lipid-modifying enzyme, SK is quite hydrophobic so I think it just tends to drop out of solution when you are trying to get the crystals. However, while that is ongoing, we are making modelled structures of the protein using in silico docking of small molecules and developing inhibitors based on that modelling.”

Pitson notes that another promising role for SK inhibitors might be in the currently hot area of chemosensitisation and resistance to cancer therapy. “Some people showed that if you target SK, not only can you suppress those tumour cells in laboratory cell culture conditions and inhibit growth in vivo, but you can also chemosensitise the cells. Some call this characteristic a non-oncogene addiction.

Essentially, this means that when you get cell transformation (normal cell into a cancer cell), SK is needed to provide extra survival signals for the tumour cells. And conversely, if you take that SK away through inhibitors or otherwise, the tumour cells not only stop growing, but also become more susceptible to the regular chemotherapeutics.”

At ComBio, Pitson will discuss the work that this group has done on SK over the last several years and particularly how CIB1 works to drive SK to the right place on the plasma membrane. He will also touch on more recent aspects of his research, including some new findings on the lesser-known sphingosine kinase, SK2.

Like the quieter younger sibling, SK2 has gone largely unstudied and uncared for in this heady explosion of work into sphingosine-related signalling over the past decade, with most of the work lavished on the showier SK1 and its tumourigenic potential. According to Pitson this is mainly because nobody really understands how SK2 works.

Also, when people first expressed SK2 in cells, they saw a growth suppression; it didn’t seem to turn normal cells into cancer cells like SK1. So, essentially it was not all that interesting in terms of potential impact on diseases like cancer.

However, Pitson’s group now have some evidence that the SK2 story is much more complex than first thought. It is unpublished as yet and so not for wider release, but suffice to say that it might just draw this other shy sphingosine kinase into the limelight as the next potential target for cancer therapy.

This feature appeared in the September/October 2011 issue of Australian Life Scientist. To subscribe to the magazine, go here.