Walsh, MarkMarkWalsh2025-11-062025-11-062024 the A2024http://hdl.handle.net/10197/29688The hypoxic tumour microenvironment is a key driver of cancer progression, eliciting a profound metabolic reprogramming that promotes cancer cell (CC) dissemination to secondary sites, where one or more multiple tumours can be formed through a multistep process known as metastasis. Malate dehydrogenase 2 (MDH2) is a key mitochondrial enzyme essential for functional TCA cycle, electron transport chain (ETC) support and is part of the malate/aspartate shuttle (MAS). This shows its vast involvement in energy production and key metabolic pathways. Publicly available data shows that MDH2 is consistently amplified in tumours, suggesting a link with malignant progression; however, its role in hypoxic metastatic cancers is still poorly understood. Stemming from this background, in this study, we hypothesised that targeting MDH2 disrupts mitochondrial metabolism and thus favours O₂-independent metabolic pathways affecting hypoxic CC metastasis. In order to delineate the role of MDH2 in the hypoxic metabolic rewiring of metastatic cancers, PANC-1 (exocrine pancreas), MDA-MB-231 TGL (breast) and MDA-MB-231 LM2 (metastatic breast cancer variant) CCs were transfected with lentiviral vectors encoding shRNAs targeting MDH2. Flow cytometry was used to determine cell cycle distribution and RT-qPCR to determine the expression levels of metabolic and pro-metastatic genes under hypoxia. Administration of MDH2 cell permeable substrate, di-methyl malate (DMM), was used to assess the role of malate accumulation in hypoxic CC proliferation, whereas polarography determined O2 consumption rate (OCR) in CCs treated with DMM. Migration and clonogenic assays were performed to assess migratory and survival abilities in hypoxic and post-hypoxic CCs, respectively. In line with the hypothesis, we found that MDH2 loss-of-function (LOF) reduced the expression levels of CA9 and HMOX1, two genes involved in hypoxic metabolic reprogramming, while decreasing PKM2 and increasing PDK1 gene expression indicating enhanced glycolytic metabolism. Furthermore, MDH2 LOF increased DNA synthesis and impaired G2-phase entry under prolonged hypoxia (1% O2). DMM reduced OCR, a finding consistent with the role of MDH2 as a key enzyme fuelling mitochondrial respiration. Functional assays show that MDH2 LOF impairs breast CC migration while reducing pancreatic CC survival under both hypoxic and post-hypoxic conditions, respectively. Our study identifies that amplification of MDH2 is a possible pathogenic driver for cancer progression and mortality, highlighting its critical role in hypoxic metabolism of metastatic cancers. Our findings suggest that targeting MDH2 could disrupt key metabolic pathways essential for steps of the metastatic cascade and offer new adjuvant therapeutic strategies in the fight against metastatic cancers.enMDH2HypoxiaMetastasisCancerDoctoral Thesishttps://creativecommons.org/licenses/by-nc-nd/3.0/ie/