Implications of stress-induced dsRNA in melanoma in the context of activating MAPK mutations in vitro/ex vivo

Abstract

Many studies in the past 10 years have drawn attention to the presence of endogenous RNA structures that can adopt double strand form (Dhir et al., 2018). These dsRNA have been proposed to play important biological roles in the pathogenesis of many inflammatory and autoimmune diseases as well as cancers (Hur, 2019a). The exact trigger for the forma4on of these dsRNA has yet to be identfied. Moreover, there is no direct method to quantify and locate dsRNA inside cells. This thesis will discuss the dsRNA stress response in melanoma in vitro/ex vivo. DsRNA derived from repetitive DNA elements has the potential to induce an interferon response that renders resistant melanoma sensitive to immunotherapy. As a consequence, dsRNA has the potential to add a new approach to treating melanoma. Tissue culture and molecular biology were used to induce dsRNA formation in melanoma cell lines using hypomethylating drugs and other stressors. The impact was measured by quantifying cytosolic dsRNA sensors, also known as pattern recognition receptors PRRs (pPKR, MDA5, RIG1, and ADAR1) using RT-qPCR and western bloVng. Immunofluorescence and high- resolution microscopy were then applied to investigate the co-localisation of dsRNA and its sensors in melanoma cell lines and melanoma patient skin biopsy samples. Moreover, a novel method of labelling dsRNA by flow cytometry to investigate its effect on the cell cycle and proliferation in BRAF mutant and WT cell lines was performed. Analysis revealed that inducing endogenous dsRNA significantly activates PRRs and promotes subcellular co-localisation with dsRNA and the mitochondria. FACS experiments indicated a potential role of dsRNA and PKR in regulating the cell cycle and proliferation. To summarize, our data support the hypothesis that inducing endogenous dsRNA activates dsRNA sensors and triggers innate immune signalling. Hence, dsRNA signalling may be explored as a potential therapeutic strategy to treat resistant melanoma.