Targeting PPP1R15 in malignancy
Lead Research Organisation:
University of Cambridge
Department Name: Cambridge Institute for Medical Research
Abstract
The goal of cancer therapy is to kill malignant cells with no or minimal harm to normal tissue. This can be achieved by identifying and targeting features specific to the cancer cell. A recurring theme in cancer biology is the inactivation of processes that would normally cause cancer cell death. Strategies that reactive these processes are therefore promising targets for the development of new treatments. Because cancer cells commonly make high levels of protein they are prone to accumulating incorrectly synthesised (so called 'misfolded') protein. The accumulation of misfolded protein in an a part of the cell called the endoplasmic reticulum (ER) leads to a toxic state called 'ER stress'. This reduces protein synthesis in the cell and activation of protective processes following the triggering of an ER stress sensor called PERK. PERK appears to be important in cancer progression because cancer cells lacking PERK form smaller tumours, while drugs that block PERK are showing great promise in research studies as anti-cancer agents. A protein called PPP1R15A normally opposes the action of PERK and we previously showed that PPP1R15A is responsible for some of the cell death caused by ER stress. Consequently, cells that have lost PPP1R15A experience more protective by PERK and we recently reported that highly aggressive forms of a cancer called malignant mesothelioma tend to lose PPP1R15A, possibly increasing the cancers resistance to ER stress.
In this project we will explore the potential for targeting PPP1R15A in the treatment of cancer. We have recently reported that PPP1R15A is regulated by the abundance of a protein called G-actin, the levels of which are sensitive to cellular growth signals and to coll movement. We will determine how the interaction between PPP1R15A and G-actin in regulated in health and whether manipulation of this interaction can increase PPP1R15A activity in the cell, since this could potentially increase the effectiveness of anti-cancer PERK inhibitors. PPP1R15A is an unstable protein that is efficiently degraded within the cells. We will identify the cellular machinery that causes PPP1R15A degradation, since inhibition of this machinery could restore PPP1R15A levels and so increase ER stress-induced cancer cell killing. Remarkably, the precise mechanism by which PPP1R15A leads to cell toxicity remains unclear. PPP1R15A can bind to cellular components including the ER, lipid droplets and mitochondria, all of which are crucial for cellular survival. By determining the effect of PPP1R15A binding on the function of these cellular structures we will better understand the toxic effects of this protein.
Together, these studies will enable us to understand the mechanism and functional consequences of PPP1R15A loss from cancer cells. This will be important in the development of anticancer therapies that target ER stress.
In this project we will explore the potential for targeting PPP1R15A in the treatment of cancer. We have recently reported that PPP1R15A is regulated by the abundance of a protein called G-actin, the levels of which are sensitive to cellular growth signals and to coll movement. We will determine how the interaction between PPP1R15A and G-actin in regulated in health and whether manipulation of this interaction can increase PPP1R15A activity in the cell, since this could potentially increase the effectiveness of anti-cancer PERK inhibitors. PPP1R15A is an unstable protein that is efficiently degraded within the cells. We will identify the cellular machinery that causes PPP1R15A degradation, since inhibition of this machinery could restore PPP1R15A levels and so increase ER stress-induced cancer cell killing. Remarkably, the precise mechanism by which PPP1R15A leads to cell toxicity remains unclear. PPP1R15A can bind to cellular components including the ER, lipid droplets and mitochondria, all of which are crucial for cellular survival. By determining the effect of PPP1R15A binding on the function of these cellular structures we will better understand the toxic effects of this protein.
Together, these studies will enable us to understand the mechanism and functional consequences of PPP1R15A loss from cancer cells. This will be important in the development of anticancer therapies that target ER stress.
Technical Summary
Deregulated protein synthesis is a feature of cancer and can lead to endoplasmic reticulum (ER) stress. A rectifying response to ER stress involves the phosphorylation of eIF2a by PERK, which inhibits new protein synthesis while triggering a gene expression programme through enhanced translation of ATF4. A target of ATF4 is PPP1R15A, which dephosphorylates eIF2a to restore normal translation. We showed that PPP1R15A contributes to cell death during unremitting ER stress by promoting accumulation of misfolded protein. Phosphorylation of eIF2a has a permissive effect on tumour growth and so PERK inhibition has emerged as a novel anticancer strategy. We observed the loss of PPP1R15A by some cancers leading us to hypothesise that restoration of PPP1R15A activity could serve to oppose PERK and potentiate the anticancer effects of PERK inhibitors.
Aim 1. Elucidate the physiological regulation of PPP1R15A: We demonstrated that PPP1R15A is regulated by the binding of G-actin. The abundance of G-actin is determined by cytoskeletal dynamics that are themselves regulated by growth and mobility cues. We will will determine how these cues areguate eIF2a phosphatase activity in health, so that we might target these pathways to augment PPP1R15A activity in disease.
Aim 2. Elucidate the regulation of PPP1R15A stability: PPP1R15A has a half-life of <4 hours. We will identify the degradation machinery responsible for this, since inhibiting PPP1R15A degradation could also promote eIF2a phosphatase activity.
Aim 3. Elucidate the role of PPP1R15 membrane binding: Binding to the ER, lipid droplets and mitochondria is a prominent but poorly understood feature of the PPP1R15 proteins. We will determine the effect such binding on the function of the organelles and on the activity and stability of PPP1R15.
By understanding the regulation, stability and membrane association of PPP1R15A we will be able to develop strategies to control its activity in diseases including cancer.
Aim 1. Elucidate the physiological regulation of PPP1R15A: We demonstrated that PPP1R15A is regulated by the binding of G-actin. The abundance of G-actin is determined by cytoskeletal dynamics that are themselves regulated by growth and mobility cues. We will will determine how these cues areguate eIF2a phosphatase activity in health, so that we might target these pathways to augment PPP1R15A activity in disease.
Aim 2. Elucidate the regulation of PPP1R15A stability: PPP1R15A has a half-life of <4 hours. We will identify the degradation machinery responsible for this, since inhibiting PPP1R15A degradation could also promote eIF2a phosphatase activity.
Aim 3. Elucidate the role of PPP1R15 membrane binding: Binding to the ER, lipid droplets and mitochondria is a prominent but poorly understood feature of the PPP1R15 proteins. We will determine the effect such binding on the function of the organelles and on the activity and stability of PPP1R15.
By understanding the regulation, stability and membrane association of PPP1R15A we will be able to develop strategies to control its activity in diseases including cancer.
Planned Impact
The main outcomes of this research will be:
(a) a new understanding of the role of PPP1R15A and the integrated stress response, the beneficiaries of which include researchers in the broad areas of cell and cancer biology as well as those researching integrated stress and related diseases (such as diabetes and neurodegenerative disorders) but especially those studying stress signalling in malignancy;
(b) extensive research training for the post-doctoral researcher, which will include molecular biology techniques, cell biology, murine models of disease and biochemistry. The practical skills as well as key transferable skills such as work planning, teamwork and project management that the post-doc will acquire will enable them to develop towards being an independent research scientist. The University of Cambridge offers additional courses on bioinformatics, information technology skills, and generic transferable skills.
(a) a new understanding of the role of PPP1R15A and the integrated stress response, the beneficiaries of which include researchers in the broad areas of cell and cancer biology as well as those researching integrated stress and related diseases (such as diabetes and neurodegenerative disorders) but especially those studying stress signalling in malignancy;
(b) extensive research training for the post-doctoral researcher, which will include molecular biology techniques, cell biology, murine models of disease and biochemistry. The practical skills as well as key transferable skills such as work planning, teamwork and project management that the post-doc will acquire will enable them to develop towards being an independent research scientist. The University of Cambridge offers additional courses on bioinformatics, information technology skills, and generic transferable skills.
Publications
Segeritz CP
(2018)
hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in a1-antitrypsin deficiency.
in Journal of hepatology
Scott RM
(2018)
Familial pneumothorax: towards precision medicine.
in Thorax
Scott R
(2018)
Familial pneumothorax: towards precision medicine.
Kubánková M
(2018)
Rotor-Based Organelle Viscosity Imaging
in Biophysical Journal
Lim R
(2018)
Time Is of the Essence: A Young Man with Recurrent Pneumothorax and Cavitating Lung Lesions.
in Annals of the American Thoracic Society
Holcman D
(2018)
Single particle trajectories reveal active endoplasmic reticulum luminal flow.
in Nature cell biology
Description | EPSRC IRC in Targeted Delivery for Hard-to-Treat Cancers |
Amount | £10,275,035 (GBP) |
Funding ID | EP/S009000/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2018 |
End | 09/2024 |
Title | FRET based endoplasmic reticulum crowding probe |
Description | Developed FRET-based system to report molecular crowding within the endoplasmic reticulum |
Type Of Material | Technology assay or reagent |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | Holcman D, Parutto P, Chambers JE, Fantham M, Young LJ, Marciniak SJ, Kaminski CF, Ron D, Avezov E. (2018). Single particle trajectories reveal active endoplasmic reticulum luminal flow. Nat Cell Biol 20:1118-1125 doi: 10.1038/s41556-018-0192-2. |
URL | https://www.nature.com/articles/s41556-018-0192-2 |
Title | Mesothelioma organoids |
Description | Mesobank is a repository of mesothelioma tissue and in vitro models. Recently, we generated new mesothelioma organoids (currently 19 models), which are 3D cell cultures that closely mimic the properties and behaviour of mesothelioma tumours found in patients. |
Type Of Material | Cell line |
Year Produced | 2022 |
Provided To Others? | No |
Impact | With Mesobank organdies, researchers can test potential therapies and develop new treatments. This resource is critical for advancing our understanding of mesothelioma and improving the lives of those affected by the disease. The use of organoids in research has the potential to greatly accelerate the pace of discovery and lead to new treatments that can help patients with mesothelioma. |
URL | https://www.mesobank.com |
Title | ROVI (Rotor based organelle viscosity imaging) |
Description | Microscopic viscosity (microviscosity) is a key determinant of diffusion in the cell and defines the rate of biological processes occurring at the nanoscale, including enzyme-driven metabolism and protein folding. Here we establish a rotor-based organelle viscosity imaging (ROVI) methodology that enables real-time quantitative mapping of cell microviscosity. This approach uses environment-sensitive dyes termed molecular rotors, covalently linked to genetically encoded probes to provide compartment-specific microviscosity measurements via fluorescence lifetime imaging. ROVI visualized spatial and temporal dynamics of microviscosity with suborganellar resolution, reporting on a microviscosity difference of nearly an order of magnitude between subcellular compartments. In the mitochondrial matrix, ROVI revealed several striking findings: a broad heterogeneity of microviscosity among individual mitochondria, unparalleled resilience to osmotic stress, and real-time changes in microviscosity during mitochondrial depolarization. These findings demonstrate the use of ROVI to explore the biophysical mechanisms underlying cell biological processes. |
Type Of Material | Technology assay or reagent |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | First method to permit comparable microviscosity measurement using the same fluorescent probe |
URL | https://pubs.acs.org/doi/10.1021/acsnano.8b00177 |
Description | Mesothelioma and ER stress |
Organisation | Royal Papworth Hospital NHS Foundation Trust |
Country | United Kingdom |
Sector | Public |
PI Contribution | We envisaged and generated a tissue microarray with which to determine if endoplasmic reticulum stress pathways are activated in mesothelioma tissues |
Collaborator Contribution | Access to archived patient tissues used in generation of tissue microarray |
Impact | This collaboration involves clinicians (including myself), scientists (from my lab) and pathologists. We have generated a tissue microarray representing over 100 individual patients and all histological subtypes of the disease. This is to be linked with a comprehensive clinical database. |
Start Year | 2010 |
Description | Cambridge Science Festival March 2019 hands-on microscopy activities for the general public (all ages) over two weekends. |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | The Cambridge Science Festival is a series of events held annually in March in Cambridge, and is the United Kingdom's largest free science festival.[1] The festival attracts more than 30,000 visitors to over 250 events.[2] University researches and students open their lecture halls and laboratories to the general public, and hold Talks, Exhibitions and Demonstrations, mostly free of charge.[1] The Marciniak lab plays a leading part in representing the Cambridge Institute for Medical Research (CIMR) in organising hands on cell biology activities. For young children these involve toys and games teaching about the structure of cells. For older children and adults, we use a variety of microscopes including fluorescence microscopy to illustrate how modern microscopy aids in the understanding of the molecular cell biology of disease. [1] "University of Cambridge Science Festival 2011". BBC News. Retrieved 21 March 2015. [2] "About - www.sciencefestival.cam.ac.uk". Retrieved 21 March 2015. |
Year(s) Of Engagement Activity | 2015,2016,2017,2018,2019 |
URL | https://www.sciencefestival.cam.ac.uk/events/grander-view-how-modern-microscopy-illuminates-disease |
Description | ISAC (Inspiring Scientists at CIMR): lab experience and mentoring event for less-advantaged Y12 pupils, 17th Feb 2023 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | ISAC/M is a four-day, non-residential work experience programme which will take place during school half term in February 2024. The first three days will be held at two University of Cambridge research Institutes (The Cambridge Institute for Medical Research; CIMR and the MRC Mitochondrial Biology Unit; MBU) which share the same building on the Cambridge Biomedical (Addenbrookes) campus. The ISAC/M programme will start at 10:00 and end at 16:00 every day from Tuesday, 20th February - Friday 23rd February. Students will take part in biomedical research and learn more about how that research can lead to new treatments. The final day of the programme (Friday 23rd February) will take place at St Catharine's College in central Cambridge, where ISAC/M students will make short presentations on the research they took part in, and obtain advice and guidance on applying to Cambridge and other universities. Lunch will be provided each day, and travel costs reimbursed for all students taking part in the programme. What will students taking part in ISAC/M do? Spend time in a research lab at either CIMR or MBU, 'shadow' researchers, learn about how research is carried out, and run an experiment Prepare a scientific presentation about the experiment they ran Attend interactive seminars to help develop scientific knowledge and skills Attend guided tours and demonstrations of scientific core facilities and see some of the different technologies used such as cutting-edge microscopes Spend an interactive day run in partnership with St Catharine's College, Cambridge with workshops on how to apply to Cambridge and similar universities, and getting guidance on personal statements Network with other placement students, admissions staff, postgraduate students, and scientists as well as attend optional mentoring slots about studying and working in research Background to the Cambridge Institute for Medical Research (CIMR) and MRC Mitochondrial Biology Unit (MBU) CIMR is a biomedical research Institute that is part of the School of Clinical Medicine at the University of Cambridge. CIMR's mission is to determine the molecular mechanisms of disease to advance human health. We focus on translation between biological research and clinical applications. The Institute has 24 research teams studying how normal biological processes inside cells can go wrong during infections, or because of rare genetic changes. By understanding these processes and the molecules involved (particularly proteins) we aim to discover better treatments for diseases and enable better outcomes for patients. |
Year(s) Of Engagement Activity | 2023 |
URL | https://www.cimr.cam.ac.uk/isacm-2024 |
Description | June Hancock Mesothelioma Research Fund 'Meet the researchers' event speaker, |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Meet the Researchers events are an opportunity for researchers and academics to engage with patients and practitioners. Non-researchers can gain insight into the aims, objectives, challenges, and opportunities of current research; researchers can hear patients' and practitioners' priorities for research. These events are designed to promote discussion and allow questions to be asked in a relaxed and informal way. The aim is to break down the barriers between researchers and patients and enable conversations that will lead to meaningful engagement and a better understanding of research. The two-hour event was opened by Dr Kate Hill, a founding Trustee and pro bono Manager of the research activity for the June Hancock Mesothelioma Research Fund (JHMRF), a campaigning charity that sponsors and promotes vital research into the causes and treatment of mesothelioma. The online format allowed the charity to open the Meet the Researchers event - previously held in-person - to a wider audience, and participants attended from across the UK and internationally. Everyone was encouraged to ask questions throughout the event and the emerging conversations between researchers, patients and practitioners were engaging and informative. |
Year(s) Of Engagement Activity | 2022,2023 |
URL | https://www.teddy.eng.cam.ac.uk/news/irc-team-meet-researchers-public-event |
Description | RareFest 2022 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Rarefest 22 was a public engagement event focused on raising awareness and promoting understanding of rare diseases. The event was held in Cambridge and aimed to bring together individuals, families, and communities affected by rare diseases, as well as healthcare professionals and researchers. The goal of Rarefest 22 was to increase public knowledge and understanding of rare diseases, and to provide support and resources to those affected. The event featured informative talks, interactive workshops, and opportunities for attendees to connect with others who have similar experiences. Several hundred members of the public attended the even held in the Guild Hall, Cambridge. |
Year(s) Of Engagement Activity | 2017,2022 |
URL | https://www.camraredisease.org/rarefest22/ |
Description | • Talk at a Mesothelioma UK patients' conference in Brighton on 5th October 2023 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | One day meeting primarily for patients and carers relating to malignant mesothelioma. |
Year(s) Of Engagement Activity | 2023 |
URL | https://www.mesothelioma.uk.com/patient-carer-day-awards-night/ |