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
Boone PM
(2019)
The Genetics of Pneumothorax.
in American journal of respiratory and critical care medicine
Bradley KL
(2021)
Role of unfolded proteins in lung disease.
in Thorax
Bravo-Pérez C
(2022)
Full-length antithrombin frameshift variant with aberrant C-terminus causes endoplasmic reticulum retention with a dominant-negative effect.
in JCI insight
Chambers JE
(2022)
Z-a1-antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state.
in Science advances
Chambers JE
(2018)
Measuring the effects of a1 -antitrypsin polymerisation on the structure and biophysical properties of the endoplasmic reticulum.
in Biology of the cell
Chambers JE
(2018)
An Optical Technique for Mapping Microviscosity Dynamics in Cellular Organelles.
in ACS nano
Dell'Anno I
(2020)
Tissue expression of lactate transporters (MCT1 and MCT4) and prognosis of malignant pleural mesothelioma (brief report).
in Journal of translational medicine
Dickens JA
(2022)
Novel insights into surfactant protein C trafficking revealed through the study of a pathogenic mutant.
in The European respiratory journal
Dickens JA
(2019)
Pulmonary endoplasmic reticulum stress-scars, smoke, and suffocation.
in The FEBS journal
Emanuelli G
(2020)
The integrated stress response in pulmonary disease.
in European respiratory review : an official journal of the European Respiratory Society
Foo CT
(2021)
Ambulatory Thoracoscopic Pleurodesis Combined With Indwelling Pleural Catheter in Malignant Pleural Effusion.
in Frontiers in surgery
Girish M
(2022)
Meta-analysis of the association between emphysematous change on thoracic computerized tomography scan and recurrent pneumothorax.
in QJM : monthly journal of the Association of Physicians
Greene CM
(2018)
Author Correction: a1-Antitrypsin deficiency.
in Nature reviews. Disease primers
Hallifax RJ
(2020)
Ambulatory management of primary spontaneous pneumothorax: an open-label, randomised controlled trial.
in Lancet (London, England)
Holcman D
(2018)
Single particle trajectories reveal active endoplasmic reticulum luminal flow.
in Nature cell biology
Kardaman N
(2022)
Catamenial pneumothorax.
in Annals of the Royal College of Surgeons of England
Kubánková M
(2018)
Rotor-Based Organelle Viscosity Imaging
in Biophysical Journal
Kubánková M
(2019)
Linker length affects photostability of protein-targeted sensor of cellular microviscosity.
in Methods and applications in fluorescence
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
Lomas DA
(2021)
Development of a small molecule that corrects misfolding and increases secretion of Z a1 -antitrypsin.
in EMBO molecular medicine
Malzer E
(2018)
The integrated stress response regulates BMP signalling through effects on translation.
in BMC biology
Marciniak SJ
(2022)
Pharmacological targeting of endoplasmic reticulum stress in disease.
in Nature reviews. Drug discovery
Marciniak SJ
(2020)
Pneumothorax and the biology of Birt-Hogg-Dubé syndrome.
in Thorax
Marciniak SJ
(2019)
Endoplasmic reticulum stress: a key player in human disease.
in The FEBS journal
Martinelli AW
(2020)
COVID-19 and pneumothorax: a multicentre retrospective case series.
in The European respiratory journal
Matsumoto K
(2020)
The Importance of Genetic Factors in the Management of Spontaneous Pneumothorax
in Current Pulmonology Reports
Obacz J
(2021)
Biological basis for novel mesothelioma therapies
in British Journal of Cancer
Ordóñez A
(2021)
Cargo receptor-assisted endoplasmic reticulum export of pathogenic a1-antitrypsin polymers.
in Cell reports
Patel V
(2019)
Inactivation of Ppp1r15a minimises weight gain and insulin resistance during caloric excess in female mice
in Scientific Reports
Rojas-Charry L
(2020)
Susceptibility to cellular stress in PS1 mutant N2a cells is associated with mitochondrial defects and altered calcium homeostasis.
in Scientific reports
Scott RM
(2018)
Familial pneumothorax: towards precision medicine.
in Thorax
Segeritz CP
(2018)
hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in a1-antitrypsin deficiency.
in Journal of hepatology
Shamseddin M
(2021)
Use of preclinical models for malignant pleural mesothelioma.
in Thorax
Smolinska A
(2021)
The SARS-CoV-2 viral load in COVID-19 patients is lower on face mask filters than on nasopharyngeal swabs.
in Scientific reports
Straková K
(2020)
HaloFlippers: A General Tool for the Fluorescence Imaging of Precisely Localized Membrane Tension Changes in Living Cells.
in ACS central science
Sutherland WJ
(2021)
A solution scan of societal options to reduce transmission and spread of respiratory viruses: SARS-CoV-2 as a case study.
in Journal of biosafety and biosecurity
Thiebaut AM
(2019)
Proteostasis During Cerebral Ischemia.
in Frontiers in neuroscience
Woodford MR
(2022)
Seventh BHD international symposium: recent scientific and clinical advancement.
in Oncotarget
Ye S
(2020)
Exploring High Aspect Ratio Gold Nanotubes as Cytosolic Agents: Structural Engineering and Uptake into Mesothelioma Cells.
in Small (Weinheim an der Bergstrasse, Germany)
| 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 | 10/2018 |
| 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 | 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/ |