Lorne 2012: Flipping cancer’s switch

It seems the more we learn about fighting cancer, the more the cancer cells learn about resisting. But the fight is far from over, with researchers like Dr Nikki Verrills working on finding new ways to ‘re-arm’ a key signalling switch that gets turned off in cancer cells, enabling them to proliferate.

Verrills is a Cancer Institute NSW Early Career Researcher Fellow at the University of Newcastle where her research group studies signalling in cancer. Specifically, she aims to understand how protein phosphatases act as tumour suppressors in cancer progression and chemotherapy resistance, with a focus on breast cancer and leukaemia.

Verrills has always been interested in the area of resistance to chemotherapy, starting with her PhD work in the area of childhood leukaemia, where she used a protein biochemistry and proteomics approach to compare proteins between patients who responded to chemotherapy and those that did not.

Verrills then moved to Newcastle for postdoctoral work and to a lab that was interested in phosphatases in asthma and neuroscience. Coming from the cancer field and knowing of the link between signalling and cancer, she was keen to marry the two areas together.

At the Lorne Conference on Protein Structure and Function in February, Verrills will describe recent and exciting work on her protein phosphatase of choice, PP2A, and how reactivating it in some leukaemia patients may block the cancer’s progress, particularly those leukaemias that respond poorly to current treatments.

Protein phosphatases are signalling enzymes that specifically remove phosphate groups from cellular proteins to modulate their function (dephosphorylation). Phosphatases usually team up with their opposing-acting enzyme, the protein kinases, in a phosphorylation/dephosphorylation mechanism that is critical to regulate all sorts of cellular functions including growth, metabolism and cell death.

Dysfunction of these mechanisms are also implicated in many abnormal cell processes such as the transformation of a normal cell into a cancerous one and the sneaky tricks that cancer cells develop to resist the effects of chemotherapy.

“Over the past 15-20 years, a huge amount of research has been done looking at these signalling pathways in cancer cells, although most of that centred on the protein kinases that do the phosphorylating,” says Verrills.

“From that, we know that these enzymes are extremely important in cancer development and, indeed, some kinases are now targets for many molecularly targeted anti-cancer drugs. There has been a lot less done on the phosphatases, but from research in the past decade it seems that to get certain cancers going you need not only overactivation of the protein kinases but, at the same time, inactivation of the phosphatases – hence their classification as tumour suppressors.”

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According to Verrills, it is not yet completely understood just where and when it is in the cancer process that this signalling imbalance is most prominent, but she can say that the phosphatase inactivation certainly seems to be important for cancer development.

For example, her lab showed that inhibiting PP2A in cultured cells, either using a pharmaceutical compound or by modifying the cells to turn off the PP2A activity, will cause cell transformation, with normal cells displaying cancer cell-like properties. In addition, work from other labs shows that mice treated with an inhibitor of PP2A will develop tumours. So, inhibiting that particular enzyme seemed to induce cancer formation.

Interestingly, Verrills also noted that the tumourigenic effect doesn’t appear to be a standard-type situation where a mutation in the gene that encodes the PP2A protein is the causative factor. “Some cancer-associated mutations in PP2A have been reported, but none of a high frequency. Rather the effect seems to be regulated strictly at the protein level by inhibiting the enzyme function.”

Flicking the switch

In terms of PP2A and treatments for cancer, Verrills therefore has to think about reactivating the enzyme in cancer cells, unlike with the protein kinases, which need to be inhibited. To this end, a big part of the team’s research centres on finding and optimising compounds that can activate this PP2A enzyme and then on working out exactly how those agents might work at the molecular level.

The ultimate aim, of course, is to develop better drugs to use therapeutically. “We know that reactivating a tumour suppressor clinically is a bit more difficult than inhibiting a protein, but we think it can be done in this case.”

At Lorne, Verrills will first touch on some recently published work from her group on the inhibition of PP2A activity in samples and cell lines from patients with acute myeloid leukaemia (AML) and on what they think might be happening in these patients. She will then concentrate on some promising findings with the pharmacological compounds that her group has been using to reactivate PP2A.

AML, which usually occurs in patients over the age of 60, is the most common type of leukaemia diagnosed in Australia. Unfortunately, it is also the most difficult to treat, prompting a push for novel and more effective therapies.

According to Verrills, five to 10 percent of AML patients have mutations in a receptor tyrosine kinase called c-KIT (also known as CD117). Although c-KIT mutations are mostly seen in AML patients who initially respond well to treatment, they are often associated with a higher relapse rate and reduced survival.

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Following the hypothesis that PP2A might be regulated by c-KIT, Verrills and her team sought to better understand the role of PP2A downstream of c-KIT and what could happen when that c-KIT function is disturbed. In a nice series of experiments, they found that activation of c-KIT in the presence of its oncogenic mutation inhibits the activity of PP2A.

“So, you need that mutation in the kinase protein for the leukaemia to occur, but importantly, you also need the downstream inhibition of PP2A. Indeed, if we reactivate PP2A, either genetically or pharmacologically, we get rid of the leukaemia.”

The study involved using mouse myeloid cell lines in culture, which were modified using viral vectors to express the appropriately mutated c-KIT protein, which is the one known to induce leukaemia, with and without PP2A reactivation.

They then took those cells and did a whole lot of analysis, including PP2A and c-KIT activity, gene and protein expression, cell viability testing, apoptosis assays and tumourigenic phenotype.

Unique target

Finally, a tumour xenograft model in mice was set up to show in vivo inhibition of cancer cell growth when PP2A was activated. The study showed that inhibition of PP2A is a crucial mediator of c-KIT tumourigenesis in AML patients.

It therefore follows that the specific activation of PP2A could provide a unique therapeutic target in treatment-resistant leukaemias expressing mutant c-KIT.

“One important point to note with all of this is that PP2A is an incredibly complex enzyme. It can consist of three different subunits and each of those has multiple possible isoforms. This coverts to about 75 different potential complexes that can be formed as the result of gene expression,” says Verrills.

“So, we have spent a lot of time teasing out the expression of all the individual subunits to see if that is how the activity is being inhibited, and it certainly seems to be part of the story because we see decreased expression of a couple of specific isoforms.”

One of the PP2A-activating compounds that Verrills and her group have been testing in their in vitro and in vivo systems has a healthy head start in terms of the pathway to clinical use.

This Novartis compound, with the catchy name of FTY720 (trade name Gilenia), has already been approved by the FDA as an immunosuppressant for use in patients with multiple sclerosis. Interestingly, reactivation of PP2A with FTY720 in the AML experimental models also induces apoptosis and inhibits c-KIT-mediated growth and survival.

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Verrills believes that they can improve on the anti-cancer properties of FTY720 and they have started developing analogues of the drug designed to activate PP2A without suppressing the immune system. As a part of that project she has gone back to her old proteomics stamping ground to try and work out the mechanisms of action of their growing bank of candidate compounds.

“We would really like to identify the exact drug target of these FTY720 analogues inside the cell that is needed to activate PP2A,” she says. Nailing this will be great for the patients of course but, according to Verrills, it is also critical in terms of getting commercial interest in any promising compounds that they find.

In ongoing work, Verrills is extending their findings with the c-KIT mutation and PP2A into other AML patients who have mutations in different types of receptor tyrosine kinases. Specifically, another kinase protein called FLT-3 is mutated in about a third of all AML patients.

At Lorne Verrills will also mention some unpublished work looking at patient samples from individuals that have either wild-type or mutated FLT3. “Again, we are looking at the activity of PP2A in those patient cells and treating those cells with our activating drugs to see if we can induce apoptosis,” she says.

In terms of other phosphatases, at the moment we are just concentrating on PP2A. With such a complex enzyme that has a huge number of different functions in normal cells, just looking at this one is certainly enough to keep us busy! It is also the hot topic at the moment in terms of a tumour suppressor phosphatase, and that helps.”