Investigating Activation of the cGAS STING Pathway in Primary and Metastatic Uveal Melanoma

Mitchinson, Katie and Fielding, Andrew and Unterholzner, Leonie (2025) Investigating Activation of the cGAS STING Pathway in Primary and Metastatic Uveal Melanoma. Masters thesis, Lancaster University.

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Abstract

Centrosome amplification (CA) is a common feature in many cancers and can act as a double-edged sword, both increasing oncogenic phenotypes and acting as a potential Achilles’ Heel. Cancer cells can use a process called centrosome clustering to survive but this can also promote genomic instability, leading to micronuclei (MN) formation. These MN can activate the Cyclic GMP-AMP Synthase, Stimulator of Interferon genes (cGAS-STING) pathway, promoting an innate immune response, whose canonical function is to recognise cytoplasmic DNA and trigger a downstream inflammatory response. Some cancers may downregulate or alter cGAS-STING signalling to evade recognition by the immune system, yet the relationship between CA and the innate immune system remains under-explored. Therefore, this project explores the relationship between CA, MN and the cGAS-STING pathway in primary and metastatic uveal melanoma (UM). Confocal microscopy revealed that there was increased MN formation in metastatic UM cells, with MN exhibiting DNA damage. Interestingly, both primary and metastatic UM cells contained MN that were devoid of cGAS at baseline and after DNA transfection. Using western blotting we showed that expression of other DNA sensing components including STING and interferon gamma Inducible protein 16 (IFI16) were variable and cell line-dependent, with STING only being found in primary UM. Further use of confocal microscopy and Enzyme Linked Immunosorbent Assay (ELISA), demonstrated a lack of downstream signalling activation in both cell lines, indicated by impaired Nuclear Factor Kappa- Light-Chain Enhancer of Activated B Cells (NF-κB) and Interferon Regulatory Factor 3 (IRF3) translocation and minimal secretion of Interleukin-6 (IL-6) or C-X-C motif chemokine ligand 10 (CXCL10) in response to DNA transfection or etoposide treatment. However, these cells retained responsiveness to RNA transfection, suggesting a specific suppression of DNA-sensing pathways. Our findings indicate that UM cells, particularly those with high levels of CA and genome instability may evade immune detection by suppressing the cGAS-STING pathway, but the extent of this suppression may vary dependent on the levels of genome instability, warranting further investigation. These findings have implications for emerging therapeutic strategies and highlight the need to further investigate innate immune pathway modulation in UM.