By blocking a widespread enzyme, Centenary researchers have shown they can slow down the movement of cells and potentially stop tumours from spreading and growing.
Using a new super-resolution microscope they’ve been able to see single molecules of the enzyme at work in a liver cancer cell line. Then they’ve used confocal microscopes to see how disrupting the enzyme slows the cells down in living cancer cells.
The enzyme is DPP9 (dipeptidyl peptidase 9) which the researchers at the Centenary Institute and the Sydney Medical School were first to discover and clone, in 1999. Ever since they’ve been studying what it does, with a view to its possible use as a cancer drug target.
“It was exciting to be able to watch the enzyme at work and then block DPP9, and see the cells slow down,” says A/Prof Mark Gorrell from Centenary’s Molecular Hepatology unit. “This gives us our clearest evidence yet that this enzyme will be a good cancer drug target.”
“What this work has shown us is that this enzyme is absolutely critical to cell movement, and without cell movement, tumors can’t grow or spread,” says Gorrell of the work, published in the leading European cell biology journal BBA Molecular Cell Research.
Using the recently acquired super-resolution microscope, Ms Hui (Emma) Zhang—one of Gorrell’s PhD students— determined where individual fluorescently tagged DPP9 molecules were located inside cells. She found that DPP9 lies on the microtubules that play a significant role in intracellular transport and in cell migration.
When cells were stimulated to move, Zhang discovered DPP9 accumulates at the leading edge of the moving cell. DPP9 was also associated with the adhesion protein complex that glues the cell to the external matrix though which it moves, acting as an anchor point to pull the cell along. When the action of DPP9 was inhibited in cells, such movement and adhesion diminished.
“DPP9 is looking more and more like a cancer drug target. But at present we have no specific inhibitors for it, even though chemists have been trying for some years to make one.” he said. “We need to throw more resources at this problem.”
During the past 15 years, Gorrell has been unveiling the properties of DPP9, which belongs to a small family of four enzymes specialised in cleaving other proteins. Members of this family modify and regulate proteins for many important functions inside and outside of cells. DPP4, for instance, is already the basis of a leading drug treatment for diabetes. DPP4 inhibitors are worth about $6 billion a year and comprise about a quarter of the diabetes drug market.
“The roadblock to developing a specific inhibitor for DPP9 has been that it is very similar physically, but not functionally, to DPP8. It has been hard to distinguish between the two chemically,” Gorrell says. He is now working on determining and publishing differences between the two enzymes, which should help chemists target their efforts better.
“This is the first paper to be generated using this new microscope, which we acquired in collaboration with Sydney University with the help of the Ramaciotti Foundation,” the Executive Director of the Centenary Institute, Prof Mathew Vadas AO says. “It is a great illustration of the value of the latest microscope imaging technologies to medical research.”
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Dipeptidyl Peptidase 9 subcellular localization and a role in cell adhesion involving focal adhesion kinase and paxillin
Dipeptidyl Peptidase 9 (DPP9) is a ubiquitously expressed member of the DPP4 gene and protease family. Deciphering the biological functions of DPP9 and its roles in pathogenesis has implicated DPP9 in tumour biology, the immune response, apoptosis, intracellular epidermal growth factor-dependent signalling and cell adhesion and migration. We investigated the intracellular distribution of DPP9 chimeric fluorescent proteins and consequent functions of DPP9. We showed that while some DPP9 is associated with mitochondria, the strongest co-localization was with microtubules. Under steady state conditions, DPP9 was not seen at the plasma membrane, but upon stimulation with either phorbol 12-myristate 13-acetate or epidermal growth factor, some DPP9 re-distributed towards the ruffling membrane. DPP9 was seen at the leading edge of the migrating cell and co-localized with the focal adhesion proteins, integrin-β1 and talin. DPP9 gene silencing and treatment with a DPP8/DPP9 specific inhibitor both reduced cell adhesion and migration. Expression of integrin-β1 and talin was decreased in DPP9-deficient and DPP9-enzyme-inactive cells. There was a concomitant decrease in the phosphorylation of focal adhesion kinase and paxillin, indicating that DPP9 knockdown or enzyme inhibition suppressed the associated adhesion signalling pathway, causing impaired cell movement. These novel findings provide mechanistic insights into the regulatory role of DPP9 in cell movement, and may thus implicate DPP9 in tissue and tumour growth and metastasis.
Hui Zhang, Yiqian Chen, Carol Wadham, Geoffrey W McCaughan, Fiona M Keane and Mark D Gorrell – Centenary Institute and Sydney Medical School, University of Sydney.
Emma Zhang used equipment acquired with assistance from the Rebecca L Cooper Medical Research Foundation, the Australian Government, The University of Sydney, Cancer Institute NSW, Perpetual Trustees and The Clive and Vera Ramaciotti Foundation.
About Mark Gorrell
Associate Professor – Centenary Institute of Cancer Medicine & Cell Biology
Group leader – Molecular Hepatology Group
Mark D. Gorrell is devoted to understanding the functions of the dipeptidyl peptidase IV [DPP4] gene family enzymes and the molecular basis of chronic liver disease and how to exploit this knowledge to alleviate human illness. DPP4 is the target of a successful new type 2 diabetes therapy that is benefiting millions of patients. Improving understanding will lead to improved therapy for chronic liver disease sufferers, whether their illness is hepatitis B or C, autoimmune disease or fatty liver disease. Authorship: 118 publications including 5 patents, with about half the publications involving the dipeptidyl peptidase 4 gene family and most of the remainder on liver disease pathogenesis. Mark trained at Australian National University [BSc, PhD], University of Melbourne School of Veterinary Science and Johns Hopkins University School of Comparative Medicine and Department of Neurology before joining Royal Prince Alfred Hospital then Centenary Institute and Sydney Medical School.
About the Molecular Hepatology Group
Molecular hepatology research is focused upon understanding the functions and functioning of enzymes of the dipeptidyl peptidase IV [DPPIV; DPP4] gene family and harnessing that knowledge to improve human health, particularly liver diseases and disorders. In addition, we work with the Pharmacy Faculty at the University of Sydney on ways to better understand and use the liver/brain enzyme Kynurenine Aminotransferase 1 [KAT-1].
About the Centenary Institute
The Centenary Institute of Cancer Medicine and Cell Biology is one of the leading independent medical research institutes in Australia. At Centenary we strive for creativity and excellence in discovering improved diagnostics, treatments, and cures for cancer, inflammatory and cardiovascular diseases.
The breadth of our translational impact is demonstrated by the extensive range of research covered – from basic to clinical and public health