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.

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.

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.

Publications

10 25 50
 
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 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 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