Organ growth, warts and all

Dr Kieran Harvey from the Laboratory of Cell Growth and Proliferation at the PeterMac Centre in Melbourne is interested in how organ growth and size are regulated during development by the newly discovered Salvador-Warts-Hippo (SWH) signalling pathway. Activation of this pathway restricts organ size by limiting cell growth and proliferation and by stimulating cell death via apoptosis.

Harvey's primary interest is how this system works in Drosophila melanogaster, where the pathway was identified, but the work also has important implications for human tumorigenesis as control of proliferation and cell survival are also central to the development of cancer.

At the Hunter Cell Biology meeting this week, Harvey will present his group's latest findings on this important signalling cascade in fruit flies and how this might translate to human disease.

Harvey's interest in the pathway with the eclectic name is more than academic - he was part of its discovery. After completing a PhD in cell biology with Sharad Kumar at the University of Adelaide in 2000, Harvey took up a five-year postdoctoral position at the Massachusetts General Hospital Cancer Center in Boston and the University of California, Berkeley. In the laboratory of developmental cell biologist Iswar Hariharan, Harvey started working with Drosophila as a model organism for the first time.

"We were screening flies for genes that gave cells a growth advantage, looking for outgrowth of tissues such as eyes and wings," Harvey says. The group identified two components of a previously undiscovered pathway - a novel gene called Salvador, and a previously known gene called Warts (published in Cell, 2002).

They subsequently found Hippo and Yorkie, and together, these four genes comprise the core components of the Drosophila SWH pathway, required for normal cell growth, proliferation and apoptosis.

Identifying Hippo and showing that it controlled tissue growth with Salvador and Warts comprised a second Cell paper for Harvey in 2003. Deregulation of the SWH pathway causes a significant and abnormal increase in organ size, which is lethal for developing flies and clearly of more general biological significance.

Twelve components of the SWH pathway have now been identified in flies, mostly using clonal screens for genes that affect organ size. All of these have mammalian counterparts and several have been implicated as tumour suppressors or oncogenes.

According to Harvey, many of these were known genes, discovered in the 1980s and 90s by different groups and later slotted into the SWH pathway. The components belong to a range of protein classes: kinases (Hippo and Warts and Discs overgrown), scaffold molecules (Salvador), membrane-associated signalling proteins (Expanded and Merlin), and cytoskeletal motors (Dachs) to transcriptional regulators (Yorkie).

---PB--- Fat and tissue growth

In early 2006, Harvey moved back to Australia to set up a group at the Peter MacCallum Cancer Centre and continue his work on Salvador-Warts-Hippo signalling. "Our major focus at the time was to fill out the pathway [as only five components were then known]," Harvey says.

"Initially we looked for a transmembrane receptor for the pathway that might signal to one or more of the so-far all intracellular components." Taking a candidate approach, Harvey looked for plasma membrane proteins previously shown to regulate tissue growth in Drosophila and came across Fat, an atypical member of the cadherin family of adhesion proteins.

According to Harvey, mutations in Fat were first discovered in flies in the 1920s with dominant alleles that affected wing size and abdomen shape.

"Then, in the late 1980s, Fat was shown to regulate tissue growth and cell proliferation, before cloning of the gene in the 90s identified it as a cadherin, but still no one knew how it worked to regulate organ development."

Cadherins span the membranes of adjacent cells to stick them together and form a barrier, but are also important for signalling to intracellular pathways.

In the first part of his presentation at the Hunter meeting, Harvey will discuss how they identified Fat as a component of the SWH pathway. "Based on earlier work by others, we did genetic studies in our flies and also stained Fat-mutant tissues for different target genes of the SWH pathway such as cyclin E, which drives cell proliferation, and DIAP1, which inhibits apoptosis."

There were strong genetic interactions between Fat and multiple components in the pathway, and animals lacking Fat were phenotypically similar to those with depressed SWH signalling.

Further studies revealed a potential mechanism by which Fat regulates SWH pathway activity. It seemed that Fat regulates the apical membrane localisation of an intracellular protein called Expanded, another upstream regulatory protein in the pathway associated with the plasma membrane.

"We are not sure at the moment how all this happens," Harvey says. "Expanded sits at the apical membrane of developing cells and presumably acts as some sort of bridge between the surface proteins and either the actin cytoskeleton or downstream signalling proteins. We therefore think that Fat restricts organ size by stimulating downstream SWH pathway components to limit transcription."

The current goal of Harvey's group is to nut out the entire SWH pathway. "We now want to know exactly how Fat signals to the downstream components, but also how Fat itself is regulated. Evidence is mounting in both Drosophila and mammals that the SWH pathway is controlled by cell adhesion, but we do not know how.

"Fat is a likely candidate to mediate adhesion dependent-signalling given that it is a cadherin, but other proteins might also be involved, such as another cadherin, Dachsous."

---PB--- Fat and Expanded

At the Hunter meeting, Harvey will also discuss new findings on the temporal control of SWH pathway activity throughout Drosophila development.

"To date, all pathway components were thought to function throughout development in flies. However, we have found that although the four core components of the pathway act early on to regulate growth and proliferation and later on in development to drive apoptosis, other upstream components act differentially.

"In particular, Fat and Expanded appear to control organ size during the growth phase, but play no role in triggering apoptosis once the organ reaches critical size."

Harvey's group has also started to look at the role of SWH pathway components in different human cancers, and several members have been implicated already. According to his recent article in Nature Reviews Cancer (Harvey and Tapon, 2007), "evidence from patient samples, cancer cell lines and mouse models indicate that disruption of the analogous human pathway is involved in tumorigenesis".

At the Peter MacCallum Cancer Centre, Harvey has access to a large array of tumour samples from patients across the cancer spectrum. Using this resource, his team will search for mutations in individual pathway genes, as well as staining tumours for expression of transcriptional enhancer protein, YAP, which is the mammalian orthologue of the fly protein yorkie.

"Essentially, instead of going in and sequencing all known pathway components in each sample, we are looking for expression of this common key downstream protein," Harvey says. "All of the upstream components of the pathway impinge on this one oncoprotein and regulate its stability, phosphorylation and shuttling between nucleus and cytoplasm."

Once his team has found a class of tumours with increased YAP expression, they will sequence the tumour suppressor genes involved and do functional assays.

Career development

Kieran Harvey received funding from the Human Frontier Science Program (HFSP) in 2006 to set up his laboratory in Australia. HFSP is a prestigious international granting body that has Australian membership through the National Health and Medical Research Council (NHMRC), involving more than $70 million in grants and fellowships each year.

Harvey also won a Career Development Award from the Leukemia Lymphoma Society and a Peter MacCallum Cancer Centre Junior Investigator Award to help kick-start his independent research career back in Australia.

As testament to those funding decisions, Harvey has continued to make pivotal findings in the field of development cell biology, and last year secured a four-year Australia Career Development Award Fellowship from the NHMRC.