Cancer is the second leading cause of death in the World. In an effort to find alternative cancer drugs, Professors P. Andrew Evans, John Allingham and Andrew Craig at Queen’s University, Canada, have developed a synthetic analogue of a natural toxin present in a marine sponge. Both the natural toxin and the team’s analogue have been found to disrupt actin filaments that allow cancer cells to move. Without actin filaments, cancer cells lose the ability to invade healthy tissues, which halts cancer metastasis, a process responsible for 90% of cancer-related deaths.
Cancer is the second leading cause of death in the World, claiming 10 million lives each year. In 2020, 4.8 million people were diagnosed with cancer in Europe alone, which amounts to 13,000 people every day, 546 every hour, 9 every minute. While cancer has always been a challenging medical condition, Dr Hand Kluge, the regional director for the European World Health Organization, has stated that a cancer epidemic is predicted for the coming years, with more cancer cases and worse patient outcomes being expected.
The challenges of cancer treatment
One of the main challenges in attempting to treat or cure cancer is that each cancer is different from all others. While there are strategies which have been found to be more effective against a certain type of cancer – skin, lung, breast cancers, and so on – each person will react differently to treatment and patient outcomes are highly dependent on the individual’s response. Frequently, a combination of different cancer therapies is found to be most effective and, as such, it is important to have access to a wide range of potential treatment options.
Despite its high variability between individuals, there is one process that is common across cancers, which leads to worse patient outcomes – metastasis. Metastasis takes place when a malignant tumour cells invade surrounding tissues, and get into the bloodstream to travel through the body, often installing itself in organs distant to the original site. In other words, metastasis is the technical name given to the spreading of cancer through the body, and it accounts for 90% of cancer-related deaths. It is therefore imperative that metastasis is avoided or, ideally, halted altogether in cancer patients.
Halting metastasis by targeting cells’ actin cytoskeleton
Metastasis is a complex process, but actin cytoskeleton reorganization in cancer cells is known to play a critical role in this mechanism. The actin cytoskeleton consists of a complex and dynamic network of actin filaments, which helps cells maintain their shape. Metastasis is driven by rapid and dynamic assembly and disassembly of these filaments, and therefore impeding this process can halt metastasis. A number of research groups have discovered cytotoxic natural products that target actin and block actin cytoskeleton reorganization in ways that can inhibit the metastasis process.
One of the problems with these natural products is that they cannot discriminate between cancerous and non-cancerous cells, and therefore they result in generalised cell death across the body, particularly in areas where there is naturally rapid cell proliferation, such as the stomach lining, hair follicles, etc. This indiscriminate cell death is a common problem with most chemotherapy agents, and it is in fact the origin of chemotherapy side effects such as nausea and hair loss. In addition to this common challenge, the anti-actin natural products are generated in small quantities, which makes them difficult and expensive to extract in the quantities needed for use in medicine.
An example of an anti-actin natural product is Mycalolide B (MycB), which is extracted from marine sponges and has been demonstrated to inhibit the formation of actin filaments in cells. This effect of the MycB toxin suppresses proliferation and motility of cancerous cells, meaning they are less able to migrate and invade cells in other organs, i.e., metastasize. Hence, MycB successfully halts metastasis and therefore shows promise as a cancer drug. However, it takes 1.8 kg of marine sponge to extract only 89 mg of MycB, which makes the toxin a very limited resource, given the origin and slow rate of growth of the natural source. Simplified versions of MycB has been prepared in a laboratory environment, but so far these agents are not active in cells.
Mimicking the marine sponge extracts
In an effort to pursue the promising results obtained with MycB, Professors P. Andrew Evans, John Allingham and Andrew Craig, and their teams at Queen’s University in Canada, developed a collection of MycB analogues based on the original MycB structure. The active component in these synthetic MycB analogues is based on the extended aliphatic “tail” region, which has been recognised to play an important role in destabilising actin. The team at Queen’s University synthesised a panel of more than 30 MycB analogues with different variations of this molecular tail and evaluated their action against actin.
Proteins are complex, large molecules that can exist in many forms and conformations. Actin, for example, can exist in a globular form (G-actin), or a filamentous form (F-actin). Different forms of the protein will expose different parts of it to possible chemical reactions or, in the case of actin, interactions with other actin subunits, which underlies F-actin assembly. In order for the MycB analogues to have the required effect of breaking down the cells’ F-actin cytoskeleton and halt metastasis, they should inhibit the polymerisation (the molecular interaction) of G-actin subunits, and destabilise the structure of F-actin (depolymerization). To have a performance comparable to the natural MycB extract, the analogues should demonstrate the ability to enter cells and supress motility and invasion of cells at low concentrations, which was demonstrated in breast and ovarian cancer cells for MycB itself. Notably, higher doses of MycB were also found to completely inhibit cell growth.
Most tests to evaluate the effects of MycB and its analogues utilise fluorescence, i.e., light emitted by actin molecules tagged with fluorophores, to monitor the processes of F-actin assembly or disassembly over time. Polymerisation of actin, for example, is monitored by an increase in fluorescence over 600 seconds. Microscopy techniques, on the other hand, utilize fluorescence of staining solutions to monitor cell death. In this case, cancer cells are stained with fluorescent dyes; while the cancer cells are alive, light is emitted and fluorescence observed, while as the cancer cells die because of the effect of the MycB analogue toxins, the fluorescence is increasingly lost.
Amongst the several MycB analogues the researchers produced with their newly developed synthetic route, there was one whose anti-actin performance stood out. The team found that this MycB analogue could inhibit the polymerisation of G-actin to effectively reduce the final amount of the polymerised product. Moreover, the team’s synthetic analogue was found to disrupt the fluorescence of F-actin, meaning that it can destabilise the structure of this form of the protein, which in combination with the inhibition activity represents an ideal outcome for anti-actin behaviour. In terms of its activity at a cellular level, the analogue was found to enter human cancer cells quickly, actively disrupting the actin cytoskeleton and causing cancer cells to lose motility, not being able to invade their surroundings. Importantly, the synthetic analogue was able to produce these results at low enough concentrations so as make it a viable lead for further development.
Despite the success of this analogue, further improvement is possible and the researchers intend to carry out further studies into developing new, more potent analogues. To guide their work, Professors Evans, Allingham and Craig’s teams sought a structural explanation for the performance of their promising MycB analogue. In particular, the researchers used X-ray crystallography to obtain information on the structure of both the analogue and the analogue-actin complex structure. By understanding how the analogue and the actin molecules bind to each other, the researchers can move forward by designing new analogues that can better attach to the actin protein, interacting with it in the required way so as to disrupt it. Using this information, the teams at Queen’s University are now working towards even more powerful MycB analogues that may display superior performance against cancer metastasis.
- Pipaliya, B. V., et al. (2021). Truncated Actin-Targeting Macrolide Derivative Blocks Cancer Cell Motility and Invasion of Extracellular Matrix. Journal of the American Chemical Society, 143, 6847-6854. DOI: https://doi.org/10.1021/jacs.0c12404
- Nersesian, S., et al. (2018). Effects of Modulating Actin Dynamics on HER2 Cancer Cell Motility and Metastasis. Sci Reports. 8:17243. DOI: https://doi.org/10.1038/s41598-018-35284-9
- Tanaka, J., et al. (2008). Actin-Binding Toxin ‘‘Tail’’ Wags the Dog. Chemistry and Biology Reviews. 15(3)205-7. DOI: https://doi.org/10.1016/j.chembiol.2008.02.012
- Allingham, J. S., et al. (2008). Structures of microfilament destabilizing toxins bound to actin provide insight into toxin design and activity. PNAS. (102)41:14527-32. https://doi.org/10.1073/pnas.0502089102
- Allingham, J. S., et al. (2006). Actin-targeting natural products: structures, properties and mechanisms of action. Cell. Mol. Life Sci. 63:2119–2134. DOI: https://doi.org/10.1007/s00018-006-6157-9
The team are developing new tools to target actin-driven cancer metastasis.
New Frontiers in Research Foundation, Collaborative Health Research Partnerships (NSERC/CIHR)
P. Andrew Evans is the Alfred R. Bader Chair of Organic Chemistry and a Tier 1 Canada Research Chair in Organic and Organometallic Chemistry in the Department of Chemistry at Queen’s University.
John S. Allingham is a former Canada Research Chair in Structural Biology and Professor in the Department of Biomedical and Molecular Sciences at Queen’s University.
Andrew Craig is a Professor in the Queen’s Cancer Research Institute.
P. Andrew Evans: Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada
John S. Allingham: Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario K7L 3N6, Canada
Andrew Craig: CRI 315, Queen’s University, Kingston, Ontario Canada K7L 3N6
P. Andrew Evans
T: +1 613 533-2496