The Ubiquitin and Proteasome System in Tumour Management and Drug Discovery

Dr Yi Sheng THE UBIQUITIN AND PROTEASOME SYSTEM IN TUMOUR MANAGEMENT AND DRUG DISCOVERY

Ubiquitin is a polypeptide that is tagged on to various proteins to signal a range of biological processes. The alteration of the ubiquitin system plays a pivotal role in the pathogenesis of diseases including autoimmune disorders and cancer. The process of ubiquitinylation involves a cascade of enzymes, E1, the activating enzyme, E2 (conjugating enzymes) and E3 (ligases). Characterisation of the ubiquitinylation process of key proteins that impact stem cells, immune cells and cancers is vital to identify therapeutic targets influencing the progression of autoimmune conditions and cancers. The ubiquitin system is compromised in the majority of cancers and is the focus of research by Dr Yi Sheng of York University, Canada.

Ubiquitin and Ubiquitin Conjugation Maintaining the balance in this dynamic, complex system requires Ubiquitin is a small protein that is not only the ability of enzymes to bind attached to other proteins in the cell ubiquitin to target proteins but also to signal specific biological processes. remove ubiquitin. Removal of ubiquitin This is a reversible process that is used is facilitated by ubiquitin-specific to alter the function of the proteins proteases which catalyse the separation in various ways. The process of of ubiquitin from the protein. attaching ubiquitin to another protein is termed ubiquitin conjugation, a P53 and Ubiquitylation Control of E3 ligases. These enzymes drive the highly regulated, sequential, multi-step Tumourigenesis ubiquitylation of p53 by attaching the process. small protein, ubiquitin, to p53. This P53 is a protein that is known to be a process signals that the tagged p53 Single ubiquitin molecules can be tumour suppressor. It plays a pivotal should be degraded. attached to proteins, a process known role in safeguarding the integrity as monoubiquitinylation, which is of the genome and preventing Human Malignant Cells and p53-E3 associated with various biological tumourigenesis, the development and ligases processes. However, ubiquitin itself can progression of cancer cells. P53 achieves further form polyubiquitin structures, this by preventing the proliferation of MDM2, an important human effectively chains of ubiquitin attached damaged cells, which occurs through E3-ubiquitin-protein ligase, is to an initial target protein. The length one of two key mechanisms. More overexpressed in a number of human and specific linkages used between specifically, by stopping the cell cycle malignancies, and as such, it is an the ubiquitin molecules indicate vastly (termed cell cycle arrest) or by inducing attractive target for novel cancer different biological outcomes for the programmed cell death (also known as therapies. From a potential therapy targeted protein. apoptosis). In normal cells, the level of perspective, inhibition of the E3 ligase p53 is closely regulated. Regulation is MDM2 in tumours should result in managed by a group of enzymes called an increase of p53 and subsequently

WWW.SCIENTIA.GLOBAL increased levels of p53-activated cell a number of E3 ligases associated discovered that these two E3 ligases, death in cancers that are overexpressing with p53 degradation, with the most MDM2 and Pirh2, target different sites MDM2. commonly known MDM2 protein to on the p53 ubiquitin tagging. While the identify differences as well as similarities significance of this is currently unclear, it In the past decade, the number of E3 in the structures and functions that may may offer future drug targeting options. ligases identified by researchers has be exploited to inform novel targets for increased substantially to include Pirh2, cancer drug therapies. Following the work on MDM2 and Pirh2, COPI, TOPORS and HUWE1 (also known Dr Sheng and her team conducted as Mule or ARF-BP1). This broad range of First, comparing MDM2 with Pirh2, structural and activity comparisons of E3 ligases results in greater complexity which is also prevalent in a number of the E3 ligases MDM2 and MDMX. They of the p53-ubiquitylation pathway and human cancers, Dr Sheng found that, successfully identified specific regions offers an increased number of potential similarly to MDM2, Pirh2 reduces p53 of the ring structures of these molecules drug targets in p53-dependent cancers. levels via ubiquitinylation. However, which are essential for ubiquitinylation. Pirh2 ubiquitylation occurs in a manner This information provides a potential MDM2 is found to be overexpressed which is fully independent of MDM2. route for designing MDM2 inhibitors in more than 10% of human cancers, This means that the inhibition of p53 which directly influence its E3 ligase including 40–60% of human osteogenic by Pirh2 may play an important role in activity and prevent the pivotal p53 sarcomas (bone cancers) and around tumourigenesis. ubiquitinylation in tumours. 30% of soft tissue sarcomas. A sarcoma is a malignant tumour which arises Dr Sheng and her team identified Research has revealed that the p53 from connective-type tissues including important structural differences regulators MDM2 and MDMX operate in muscle, tendons and blood vessels. between MDM2 and Pirh2. In addition, different ways, each serving to provide MDM2 is also frequently overexpressed they conducted tests to determine their a specific and unique role in regulating in the malignancies of blood cells, which respective ubiquitylation activities, both the cell death (MDM2) and cell cycle causes the inhibition of p53. autoubiquitylation – self-tagging with arrest (MDMX) actions of p53. ubiquitin – and p53 ubiquitinylation. Molecular and Functional Regulation of the Wnt Pathway Comparison of MDM2 and other Dr Sheng and her team found that E3 ligases MDM2 performs as a more effective Another pathway which is regulated by ubiquitylation enzyme for both ubiquitinylation is the Wnt pathway, Dr Yi Sheng of York University, Canada, auto- and p53-ubiquitylation than which is key in both embryonic and her colleagues have studied Pirh2. During their testing, the team development and maintenance of

WWW.SCIENTIA.GLOBAL homoeostasis in adult tissues. Two of the most important MDM2, an E3 ligase, is a negative regulator of p53, a potent pathways managed by the Wnt pathway are cell proliferation tumour suppressor protein. MDM2 promotes ubiquitylation and and stem cell self-renewal. Genetic manipulation or failed degradation of p53. modulation of the Wnt pathway contributes to cancer development in a number of tissues. In blood cell tumours, MDM2 overexpression is frequently detected where p53 is commonly inactivated through negative ß-catenin is a protein which regulates and coordinates gene regulation. Dr Sheng and her team’s current research focuses transcription, the first step in gene expression. Specifically, on identifying and developing lead compounds that inhibit the it is the intracellular signal transducer in the Wnt signalling E3 ligase activity of the tumour-promoting protein MDM2. pathway. Mutation or overexpression of ß-catenin is associated with many cancers. In turn, ß-catenin is regulated and Dr Sheng and her team have used computational destroyed by the APC protein complex. methodologies to screen a quarter of a million naturally occurring candidate compounds to determine whether they In collaboration with the Tak Mak Lab, Dr Sheng and her team’s are capable of binding the specific region of MDM2 (the RING research has demonstrated that in conditions of hyperactive domain) that is responsible for the E3 ligase activity. Wnt signalling, an E3 ubiquitin ligase (Mule) targets ß-catenin for degradation, which in turn stops the activation of Wnt The team have identified candidate compounds as an inhibitor signalling. The work demonstrated that a combined loss of of MDM2 E3 ligase activity, and subsequently activates p53 both Mule and APC promotes the conversion of some stem cells leading to programmed cell death in human bone cancers. The into cancer stem cells, initiating cancer development. team’s on-going and future work is based on understanding the effect of this molecule in terms of toxicity and efficacy when This knowledge offers a number of intervention points for tested against a panel of haematopoietic cell lines that have the manipulation of ‘Mule’, ß-catenin and the Wnt pathway been isolated from blood cancer patients. functions to impact the initiation and development of tumours. The outcomes from this planned research will contribute to the Future Research body of knowledge surrounding the molecular mechanism of the cancer loop incorporating p53-MDM2-MDMX, and will also The ubiquitin proteasome system (UPS) governs the vast provide critical information to the scientific community on the majority of cellular protein degradation. The UPS function benefits of targeting MDM2 E3 ligase activity as a therapeutic is compromised in most cancer cells. As described above, strategy.

WWW.SCIENTIA.GLOBAL Meet the researcher Dr Yi Sheng Associate Professor Department of Biology Faculty of Science York University Toronto Canada

Dr Yi Sheng is an Associate Professor of Biology in the Faculty FURTHER READING of Science at York University, Toronto, Canada. She completed her Bachelor’s and Master’s degrees at Zhongshan (Sun Yatsen) O Egorova, HHC Lau, K McGraphery, Yi Sheng, Mdm2 and MdmX University in 1996 and thereafter undertook doctoral studies RING Domains Play Distinct Roles in the Regulation of p53 at the University of Toronto, which she completed in 2003. Responses: A Comparative Study of Mdm2 and MdmX RING After completing a Postdoctoral Fellowship with the University Domains in U2OS Cells, International Journal of Molecular Health Network, she undertook an Assistant Professorship Sciences, 2020, 21, 1309. at York University and in 2013 was promoted to Associate C Dominguez-Brauer, R Khatun, AJ Elia, et al., E3 ubiquitin Professor at the same institution. Dr Sheng’s work is focused on ligase Mule targets ß- catenin under conditions of hyperactive defining the mechanisms of the ubiquitin system and its effect Wnt signaling, Proceedings of the National Academy of on cancer pathways, and includes the development of novel Sciences of the United States of America, 2017, 114, E1148– therapeutics for potential cancer therapeutics. E1157.

CONTACT C Dominguez-Brauer, R Khatun, AJ Elia, et al., E3 ubiquitin ligase Mule targets ß-catenin under conditions of hyperactive T: (416) 7362100 ext 33521 Wnt signaling, Proceedings of the National Academy of Fax: (416) 7365898 Sciences of the United States of America, 2017, E1148–E1157. [email protected] E: K Wheaton, F Sarkari, B Stanly Johns, et al., UbE2E1/UBCH6 Is http://www.yorku.ca/yisheng/ W: a Critical in Vivo E2 for the PRC1-catalyzed Ubiquitination of H2A at Lys-119, Journal of Biological Chemistry, 2017, 292(7), KEY COLLABORATORS 2893–2902.

Cheryl Arrowsmith (Princess Margaret Cancer Center, Toronto, O Egorova, Y Sheng, A site-directed mutagenesis study of the Canada) MdmX RING domain, Biochemical and Biophysical Research Tak Mak (Princess Margaret Cancer Center, Toronto, Canada) Communications, 2014, 447, 696–701. Brian Raught (Princess Margaret Cancer Center, Toronto, F Sarkari, K Wheaton, A La Delfa, et al, Ubiquitin-specific Canada) protease 7 is a regulator of ubiquitin-conjugating enzyme Vivian Saridakis (York University, Canada) UbE2E1, Journal of Biological Chemistry, 2013, 288(23), 16975–16985.

J Shloush, JE Vlassov, I Engson, et al, Structural and Functional Comparison of the RING Domains of Two p53 E3 Ligases, Mdm2 and Pirh2, Journal of Biological Chemistry, 2011, 286(6), 4796–4808.

Y Sheng, JH Hong, R Doherty, et al, A Human Ubiquitin Conjugating Enzyme (E2)-HECT E3 Ligase Structure-function Screen, Molecular & Cellular Proteomics, 2012, 11(8), 329–341.