The role of apoptotic cell morphology in anti-tumour immunity

Naylor, Gregory (2019) The role of apoptotic cell morphology in anti-tumour immunity. PhD thesis, University of Glasgow.

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Abstract

Rho associated Coiled-Coil Kinase (ROCK) plays a critical role in many cellular functions which are dependent on the cytoskeleton. ROCK mediated actomyosin contractile force generation has been shown to be important in cell migration, proliferation and phagocytosis. During apoptosis, caspase cleavage of ROCK results in a constitutively active form. This generates a more pronounced contractile force which is required to produce many of the morphological and structural changes associated with apoptosis including cell shrinkage, nuclear disintegration and membrane blebbing. Data generated from within the Olson lab suggests that cells which undergo apoptosis when ROCK is inhibited undergo a biochemically normal but morphologically aberrant form of apoptosis which is more inflammatory and can lead to an immune mediated response. Recently published data has also demonstrated the ability of ROCK inhibition to generate a more pronounced immune response to apoptosis. We wished to investigate the mechanisms underlying this immune phenomenon and to explore if any of the observed effects may be translatable as potentially clinically useful treatments.
In order to achieve this we utilised two approaches. Firstly we employed a novel genetic mouse model of ROCK1, where a single amino acid substitution in the caspase cleavage site (D1113A) that converts ROCK1 to a caspase-resistant non-cleavable (ROCK1nc) form. This was previously generated in the Olson lab and can be used to investigate the caspase cleavage specific effects of ROCK activation. Secondly we used pharmacological ROCK inhibitors fasudil, which is currently used in clinical practice, and AT13148, which has entered early phase clinical trials in humans.
When combined with the Eµ-Myc mouse lymphoma model, ROCK1nc was associated with prolonged survival and increased macrophage infiltration into bone marrow. An effect which could be reversed by inhibition of macrophage migration.
Cultured hepatocytes expressing the ROCK1nc mutation exposed to different apoptotic stimuli displayed some differences in the levels of released damage associated molecular patterns (DAMPs). However only significant changes were noted between ultraviolet treated and control cells but not ROCK1nc and ROCK1wt cells, indicating that the apoptotic stimulus, but not necessarily caspase cleavage of ROCK, plays a critical role in DAMP expression. ROCK1nc also did not appear to impact on phagocytosis of apoptotic hepatocytes, phagocyte migration or activation of phagocytic cells in vitro. Inhibition of HMGB1 DAMP signalling at the time of induction of liver cancer in a diethylnitrosamine (DEN) mouse model increased tumour burden indicating that reduction in either neutrophil mediated inflammation or HMGB1 induced hepatocyte apoptosis leads to increased tumour burden – the effects were however not specific to ROCK1nc mice compared to controls.
Pharmacological ROCK inhibition in the DEN liver cancer model reduced tumour burden, consistent with previous results seen in the ROCK1nc model, however it was not necessarily clear that an immune medicated phenomenon was responsible for this effect. Indeed following an acute inflammatory liver insult with DEN, pharmacological ROCK inhibition appeared to reduce inflammatory response. Combination of ROCK inhibition with cytotoxic chemotherapy in the DEN model also reduced tumour burden, but not to a greater extent than ROCK inhibition alone.
These results indicate that ROCK inhibition may have an important role in limiting tumour development and are consistent with other published data. An immune mediated phenomenon could not be clearly demonstrated as a driver for the effects seen in the models assessed and it may be that other effects of ROCK inhibition such as blockade of growth factor signalling pathways plays an important role. However the effects of ROCK inhibition on reducing tumour burden make ROCK inhibitors attractive candidates as potential clinically useful anticancer drugs.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Rho associated Coiled-coil kinase, ROCK, Cancer immunity, Fasudil, Hepatocellular carcinoma,
Subjects: R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
R Medicine > RM Therapeutics. Pharmacology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences > Beatson Institute of Cancer Research
Supervisor's Name: Nibbs, Prof. Rob and Olson, Prof. Mike
Date of Award: 2019
Embargo Date: 24 March 2023
Depositing User: Dr Gregory Naylor
Unique ID: glathesis:2019-80289
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Mar 2020 08:52
Last Modified: 27 Mar 2020 09:01
URI: http://theses.gla.ac.uk/id/eprint/80289

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