Exploring DJ-1 as a Therapeutic Target in 2D and 3D Models of Melanoma

Szlosarek, Emilia Apollonia (2025) Exploring DJ-1 as a Therapeutic Target in 2D and 3D Models of Melanoma. Masters thesis, University of East Anglia.

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Abstract

DJ-1, also known as PARK7, is a Parkinson’s disease-associated protein and oxidative stress sensor, which can activate the redox-sensitive Nrf2 pathway. Recent evidence has demonstrated that DJ-1 is overexpressed in various cancers, including melanoma, suggesting it has a role in cancer development and progression and may therefore serve as a potential therapeutic target. The mechanisms behind this role remain incompletely elucidated; nonetheless, it appears to be due to DJ-1’s capabilities in protecting cancer cells from oxidative damage and its regulation of multiple signalling pathways involved in carcinogenesis and chemoresistance, including the Nrf2 pathway. This project aimed to investigate the role of DJ-1 and its potential as a therapeutic target in melanoma via 2D and 3D in vitro tissue models.

In a 2D in vitro model of melanoma, metastatic A375 DJ-1 expressing cells cultured in a monolayer exhibited DJ-1 protein in both the cytoplasm and nuclei. DJ-1 knockdown via siRNA transfection was validated by siGLO and RT-qPCR, with results showing that 100 nM DJ-1 siRNA decreased DJ-1 mRNA levels by 65% but had no significant effects on Nrf2, heme oxygenase-1 or NADPH quinone oxidoreductase-1 mRNA expression, two downstream target genes of Nrf2. 100 nM DJ-1 siRNA significantly decreased A375 cell viability, suggesting that DJ-1 may influence A375 cell proliferation. Addition of the BRAF inhibitor, vemurafenib (1 μM), following knockdown had no extra effect on cell viability compared to the non-targeting control in combination with vemurafenib (1 μM).

To test DJ-1’s potential as a therapeutic target for melanoma, experiments involving the small molecule inhibitor of DJ-1, STK793590, were conducted. STK793590 targets the DJ-1 homodimer. STK793590 (50 μM) in combination with vemurafenib (1 μM) reduced A375 cell viability by 58% compared to the DMSO control. Intriguingly, STK793590 had no eaects on mRNA expression of DJ-1, Nrf2, heme oxygenase-1 or NADPH quinone oxidoreductase-1. Following experiments in the 2D in vitro model of melanoma, 3D in vitro models of melanoma were developed and optimised to test the effects of STK793590 in combination with vemurafenib on A375 tumouroid growth. The Matrigel Matrix, ‘hanging drop’ and ultra-low adherence (ULA) plate methods were investigated. Out of these, the ULA plate method proved most favourable to conduct these experiments. Vemurafenib alone (1 μM) and STK793590 (50 μM) in combination with vemurafenib (1 μM) caused significant decreases in A375 tumouroid growth, however the combination was no better than vemurafenib treatment alone. Overall, this project revealed disparate results in 2D in vitro models compared to 3D in vitro models, necessitating further studies to confirm the potential of DJ-1 in vivo as a therapeutic target in skin melanoma.

Item Type:	Thesis (Masters)
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology
Depositing User:	Chris White
Date Deposited:	26 Nov 2025 14:16
Last Modified:	26 Nov 2025 14:16
URI:	https://ueaeprints.uea.ac.uk/id/eprint/101109
DOI:

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